Method for diagnosing malignant tumors

ABSTRACT

This invention is directed to a novel group of compounds, herein termed Recognins. Recognins are made by treating malignant tumor cells or artificial cancer cells and separating the desired products. The Recognins may be used to prepare their Chemoreciprocals, i.e., by contacting the Recognins or the Recognins on a support with body fluids. These Chemoreciprocals are useful for diagnostic and therapeutic purposes, i.e., for diagnosing and treating cancers. The Chemoreciprocals are substances which react with immunochemical-like specificity with a Recognin in vivo or in vitro, e.g., in a quantitative precipitin test, in Ouchterlony double diffusion or in immunofluorescence.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.281,883, filed July 9, 1981 and application Ser. No. 271,645, filed June8, 1981, now U.S. Pat. No. 4,486,538; which in turn arecontinuations-in-part of application Ser. No. 922,799, filed July 7,1978, now U.S. Pat. No. 4,298, 590 and also a continuation ofapplication Ser. No. 19,078, filed Mar. 9, 1978, now abandoned; which inturn is a continuation-in-part of application Ser. No. 941,940, filedSept. 13, 1980, now abandoned; which in turn is a divisional ofapplication Ser. No. 852,200, filed Nov. 17, 1977, now U.S. Pat. No.4,196,186; which in turn is a continuation of application Ser. No.621,112, filed Oct. 9, 1975, now abandoned; which in turn is acontinuation-in-part of each of applications Ser. No. 553,075, filedFeb. 25, 1975, now abandoned; Ser. No. 550,432, filed Feb. 18, 1975, nowabandoned; Ser. No. 450,404, filed Mar. 12, 1974, now abandoned; andSer. No. 385,451, filed Aug. 3, 1973, now abandoned; and thisapplication is also a continuation-in-part of application Ser. No.286,172, filed July 23, 1981; which in turn is a continuation ofapplication Ser. No. 101,127, filed Dec. 7, 1979, now abandoned; whichin turn is a continuation of application Ser. No. 852,200.

This invention is directed to a novel group of compounds, herein termedRecognins. Recognins are made by treating malignant tumor cells orartificial cancer cells and separating the desired products. TheRecognins may be used to prepare their Chemoreciprocals, i.e., bycontacting the Recognins or the Recognins on a support with body fluids.These Chemoreciprocals are useful for diagnostic and therapeuticpurposes, i.e., for diagnosing and treating cancers. TheChemoreciprocals are substances which react with immunochemical-likespecificity with a Recognin in vivo or in vitro, e.g., in a quantitativeprecipitin test, in Ouchterlony double diffusion or inimmunofluorescence.

One of the Recognins of the present invention is Astrocytin. Astrocytinis produced from brain tumor tissue, preferably brain glioma tumortissue. Protein fractions containing the Astrocytin precursor are firstextracted from the tissue. A preferred method of accomplishing theextraction is to treat the tissue with a neutral buffer under conditionsof homogenization or other techniques to disrupt the cells and tissuesin order to solubilize protein fractions which contain the Astrocytinprecursor.

At this point, the Astrocytin precursor is still bound to many largemolecular weight substances including protein, glyco-proteins,lipoproteins, nucleic acids, nucleoproteins, etc. The solubilizedproteins are then separated from the resultant tissue extract. Theextract solution from the tissue is then clarified to remove insolubleparticles. The low molecular weight contaminants are then removed fromthe resultant solution, by a perevaporation concentration technique. Thesolution which is obtained is then treated to cleave Astrocytinprecursor from other contaminants in order to obtain the proteinfraction having a pK range between 1 and 4. Thus, for example, thesolution is placed on a chromatographic column and eluted withincreasingly acidic solvents. All of the fractions which are eluted inthe neutral or acid range down to pK 4 are discarded and those fractionswith pK range 1-4 are collected. The eluate is then treated to obtain aproduct having a molecular weight of about 8,000. This is accomplished,for example, by first filtering the material to remove low molecularweight substances, i.e., those below 1,000 molecular weight, andfiltering again to remove those above 25,000. The fraction having amolecular weight between 1,000 and 25,000 is then further treated, i.e.,by thin layer gel (TLG) chromatography, to obtain Astrocytin.

Thus Astrocytin may be produced by extracting brain glioma tumor tissuewith a neutral buffer, by repeated homogenization and high speedcentrifugation, separating from the resulting extract the fractionhaving a pK range of from about 1 to 4, separating from said fractionthe substances having a high molecular weight, i.e., up to about230,000, and isolating therefrom the product Astrocytin having amolecular weight of about 8,000.

The product Astrocytin prepared in accordance with this process ischaracterized by forming a single line precipitate with its specificantibody in quantitative precipitin tests and Ouchterlony gel diffusiontests, being soluble in water and aqueous solutions having an acid orneutral pH, and insoluble at an alkaline pH, having a spectrophotometricabsorption peak wave length of 280 mu and having a molecular weight ofabout 8,000.

Astrocytin is also characterized by having a very high percentage ofresidues of glutamic acid and aspartic acid and a very high ratio ofthese acids to histidine. A further analysis of Astrocytin is providedbelow.

In a manner similar to that described above, another Recognin, calledMalignin, is produced from artificial cancer cells, i.e., cancer cellsgrown in in vitro fermentation. Malignin has a molecular weight of about10,000 and similar but distinct amino acid residue composition toAstrocytin, i.e., high ratios of glutamic acid and aspartic acid andhigh ratios of these acids to histidine. A further analysis of Maligninis provided below.

Thus, Malignin can be produced by extracting artificial brain gliomacancer cells grown in fermentation culture with a neutral buffer byrepeated homogenization and high speed centrifugation, separating fromthe resulting extract the fraction having a pK range of about 1 to 4,separating from said fraction the substances having a high molecularweight, i.e. up to about 230,000, and isolating therefrom the producthaving a molecular weight of about 10,000.

The amount of Malignin produced in artificial cell fermentation and thepercentage of total protein produced in artificial cell fermentationwhich is Malignin can be increased by growth of artificial cancer cellculture in large size growth containers.

Malignin prepared in accordance with this process is characterized byforming a single line precipitate with its specific antibody inquantitative precipitin tests and Ouchterlony gel diffusion tests, beingsoluble in water and aqueous solutions having an acid or neutral pH, andinsoluble at an alkaline pH, having a spectrophotometric absorption peakwave length of 280 mu and having a molecular weight of about 10,000.

In a manner similar to that described above, another Recognin, calledRecognin M, is produced from artificial mammary cancer cells, i.e.,mammary cancer cells grown in in vitro fermentation. Recognin M has amolecular weight of about 8,000 and similar but distinct amino acidresidue composition to Astrocytin and Malignin i.e., high ratios ofglutamic acid and aspartic acid and high ratios of these acids tohistidine. The principal compositional differences between Malignin andRecognin M are that the latter has decreased amounts of aspartic acid,glutamic acid, leucine and tryosine and increased amounts of proline,glycine and alanine. A further analysis of Recognin M is provided below.

Thus, Recognin M can be produced by extracting artificial mammary cancercells grown in fermentation culture with a neutral buffer by repeatedhomogenization and high speed centrifugation, separating from theresulting extract the fraction having a pK range of about 1 to 4,separating from said fraction the substances having a high molecularweight, i.e., up to about 230,000, and isolating therefrom the producthaving a molecular weight of about 8,000.

Recognin M prepared in accordance with this process is characterized byforming a single line precipitate with its specific anitbody inquantitative precipitin tests and Ouchterlony gel diffusion tests, beingsoluble in water and aqueous solutions having an acid or neutral pH, andinsoluble at an alkaline pH, having a spectrophotometric absorption peakwave length of 280 mu and having a molecular weight of about 8,000.

In a manner similar to that described above, another Recognin, calledRecognin L, is produced from artificial lymphoma cancer cells, i.e.,lymphoma cancer cells grown in in vitro fermentation. Recognin L has amolecular weight of about 8,000 and similar but distint amino acidresidue composition to Astrocytin, Malignin and Recognin M, i.e., highratios of glutamic acid and aspartic acid and high ratios of these acidsto histidine. As compared to Malignin, Recognin L has decreased amountsof aspartic acid, glutamic acid, leucine and tryosine and decreasedamonts of proline, glycine and alamine. A further analysis of Recognin Lis provided below.

Thus, Recognin L can be produced by extracting artificial lymphomacancer cells grown in fermentation culture with a neutral buffer byrepeated homogenization and high speed centrifugation, separating fromthe resulting extract the fraction having a pK range of about 1 to 4,separating from said fraction the substances having a high molecularweight, i.e., up to about 230,000, and isolating therefrom the producthaving a molecular weight of about 8,000.

Recognin L prepared in accordance with this process is characterized byforming a single line precipitate with its specific antibody inquantitative precipitin tests and Ouchterlony gel diffusion tests, beingsoluble in water and aqueous solutions having an acid or neutral pH, andinsoluble at an alkaline pH, having a spectrophotometric absorption peakwave length of 280 mu and having a molecular weight of about 8,000.

Recognins are further characterized by being capable of complexing withbromoacetylcellulose to form bromoacetylcellulose-Recognin and producingthe specific antibodies Anti-Recognin upon injection into mammals, saidAnti-Recognin attaching specifically to the Recognin-precursor in situ.For example, one Anti-Recognin, Anti-Malignin being toxic to brain tumorcells in vitro.

Recognins, such as Astrocytin, Malignin, Recognin M and Recognin L andsimilar substances are useful as products which may be introduced into abiological system to reduce foreign reactions, such as by coating amaterial with a Recognin. A further example may be to introduce aRecognin in order to produce the Chemo-reciprocals in the biologicalsystem. They may also be used nutritionally to encourage the growth of aparticular biological system of which they are a part. A further utilityof Recognin is the production of Target regaents which comprise thecomplexes of the Recognin with a carrier to facilitate its applicabilityin biological systems. Thus, for example, the complex conveys thephysicalchemical characteristics of the Recognin itself. The carriershould be selected from those which form a complex with the Recognin andwhich are substantially biologically inert.

Any substance known in the art which will form a stable complex withpolypeptides or proteins may be useful for complexing with the Recognin.An example is a cellulose-based material, such as bromoacetyl-cellulose.In addition to being inert to the biological system, the carrier shouldbe one that does not alter the specific physical-chemical properties ofthe Recognin which are useful for the purposes set forth herein.

The complexes of the Recognin and its carrier are useful for producing,separating and identifying its chemoreciprocal in any biological systemwith which it is brought into contact. The Recognin-carrier complex isalso useful for stimulating the production of its chemoreciprocalprecursor in any biological system into which it is introduced.

One class of Chemoreciprocals are the anti-Recognins, i.e.,anti-Astrocytin, anti-Malignin, anti-Recognin M and anti-Recognin L.These may be made by injecting the Recognin into a biological system. Animmunologically effective dose of Recognin is brought into contact withbody tissues or fluids in a manner which induces an antibody response inaccordance with techniques known in the art for producing antibodies.The anti-Recognins may be used for the delivery of materials such asdiagnostic, nutritional and therapeutic agents to specific cells orsites in a biological system which comprises introducing said agent incomplexed form with the anti-Recognin into the biological system. Theanti-Recognins are also useful for diagnosing the presence of tumorcells in a histology section, by applying the Anti-Recognin conjugatedwith a labeling substance such as dyes and radioactive substances, tosaid section, whereby staining or radioactive labeling occurs only withtumor cells. Yet another use for anti-Recognins is for increasing theyield of other useful Chemoreciprocal products (such as TAG, describedbelow) from a mammal which comprises injecting an immunologicallyeffective dose of Recognin into the mammal, or other biological system.

Prior to the present invention, glycoprotein complexes were preparedfrom brain tissue and antibodies made to them. Separated materials knownas 10B glycoproteins produced from Tay-Sachs' disease brain, wereinjected into rabbits and antibodies produced. These Tay-Sachs'antibodies were used in immunofluorescent studies of brain-containingtumors: only reactive normal non-tumor glia, not tumor glia, werestained by these antibodies.

In contrast, when Astrocytin was produced from tumor tissue, andantibodies to Astrocytin (Anti-Astrocytin) were produced and employed inimmunofluorescent studies of brain, only tumor glia, not normal(non-tumor) glia, were stained by Anti-Astrocytin. Thus, by tissue oforigin and by nature of the antibody, as well as by the specific type ofcells stained, anti-Tay-Sachs' antibodies and anti-Astrocytin areclearly different products.

Another class of Chemoreciprocals is Target reagents complexed withtheir chemoreciprocals. For example, Astrocytin Target (the product ofAstrocytin complexed with a carrier such as bromoacetylcellulose) isbrought into contact with antiAstrocytin. This type of compound may becoupled with and used for the delivery of diagnostic, nutritional andtherapeutic agents to specific cells or sites in a biological system.These compounds may also be used for purification procedures. Forexample, Anti-Astrocytin may be made by the decomplexing ofBomoacetylcellulose-Astrocytin-Anti-Astrocytin by hydrolytic treatmentwith an acid or proteinase enzyme. Target reagents are also useful forincreasing the amount of TAG products (described below) in a biologicalsystem, such as by bringing an immunologically effective dose of Targetinto contact with body tissues of fluids.

Additional Chemoreciprocals are TAG reagents (Target-AttachingGlobulins). The TAG products are produced by bringing Target reagentsinto contact with body fluids for varying periods of time to form acomplex and cleaving TAG therefrom. Two useful embodiments are S-TAG andF-TAG.

A process for producing S-TAG (Slow-Target-Attaching-Globulin) comprisesreacting blood serum or other body fluid with Target (e.g.,Bromoacetylcelluose-Malignin, Bromoacetylcellulose-Recognin M,Bromacetylcellulose-Recognin L) for approximately two hours or more at alow temperature, e.g., about 4° C., and cleaving S-TAG from theresulting material, e.g., with dilute acid for approximately two hoursat a temperature of about 37° C. The product S-TAG prepared inaccordance with this process is characterized by being soluble inaqueous buffered solutions, forming a single line precipitate with itscorresponding Recognin in Ouchterlony gel diffusion tests, beingnon-dialyzable in cellophane membranes, being retained by milliporefilters which retain molecules over 25,000 molecular weight, havingmolecular weights in different states of aggregation as determined bythin layer gel chromatography of approximately 50,000, and multiplesthereof into the macroglobulin range and having a spectrophotometerabsorption peak wave length of 280 mu.

A process for producing F-TAG (Fast-Target-Attaching-Globulin) comprisesreacting blood serum or other body fluid with Target (e.g.,Bromoacetylcellulose-Malignin, Bromoacetylcellulose-Recognin L,Bromacetylcellulose Recognin M) for approximately 10 minutes at a lowtemperature, e.g. about 4° C., and cleaving F-TAG from the resultingmaterial, e.g. with dilute acid for approximately two hours at atemperature of about 37° C. The product F-TAG prepared in accordancewith this process is characterized by being soluble in aqueous bufferedsolutions, forming a single line precipitate with its correspondingRecognin in Ouchterlony gel diffusion tests, being nondialyzable incellophane membranes, being retained by millipore filters which retainmolecules over 25,000 molecular weight, having molecular weights indifferent states of aggregation as determined by thin layer gelchromatography of approximately 50,000, and multiples thereof into themacroglobulin range and having a spectrophotometer absorption peak wavelength of 280 mu.

TAG products are useful for detecting cancer tumors in living mammals bydetermining the concentration of S-TAG and F-TAG produced by a knownvolume of the mammal's blood serum or other body fluid and correlatingthis concentration with amounts determined to be indicative of cancer.TAG products are also useful for diagnosing the presence of tumor cellsin a histology section, which comprises applying TAG conjugated with alabeling substance such as dyes and radioactive substances, to saidsection, whereby staining or radioactive labeling occurs only with tumorcells. TAG products additionally have been found to be cytotoxic totumor cells. TAG products are also useful for directing the delivery ofdiagnostic, nutritional and therapeutic agents to specific cells orsites by introducing said agents in complexed form with the TAG product.

Normal cell division in plants or animals is restricted or inhibitedwhen the cells come to occupy fully a particular space. The mechanisms(a) by which normal cells "recognize" that they have filled the spaceavailable to them, and (b) by which the operation of this recognitionmechanism in turn inhibits cell division, have both been unknown. Agroup of compounds have now been produced whose precursors are increasedin concentration when normal recognition and learning occur, and whichrelate to recognition and learning in particles and cells, and with theconnection of cells to each other. These compounds are termed RECOGNINS.By attempting to produce these compounds from normal cancer cells, itwas discovered that they are absent as such, and that changes in theirmolecular structure have occurred at the same time that the cancer cellshave lost their ability (a) to recognize that they have filled theirnormal volume, and/or (b) to stop dividing when they have filled theirnormal volume.

Novel compounds and methods for producing such compounds have beendiscovered. These new compounds are termed RECOGNINS. RECOGNINS arenovel compounds which have physiochemical characteristics which mimicthose configurations characteristic of cancer cells in terms of theirfailure to recognize and stop cell division. The use of RECOGNINS goesbeyond insight into the cancer mechanism, for immediate products andmethods are thereby provided which are useful in the diagnosis andtreatment of cancer, and for its prevention.

Methods by which artificially cultured cells can be used to produceRecognins have been discovered. One advantage of the methods disclosedherein is that Recognins and new products from them can now bemanufactured efficiently in virtually limitless quantities.

This invention transcends the field of cancer research and isimmediately applicable to any and all biological systems in which it isdesired to influence all growth and metabolism. Thus by the manufactureof the particular compound or compounds of appropriate cell type inartificial culture, and the further manufacture of products from thesesubstances, specific influence may for the first time be brought to bearon any tissue, cell, cell organelle, sub-organelle molecule or molecularaggregate in any living system. Thus specific nutritional influences atcritical times in development, specific diagnostic, preventative andtreatment methods, and the construction of artificial bioelectricalsystem (as in tissue or organ transplants) can all be affected for thefirst time. These artificial bioelectrical systems can now be made tobear the characteristics of the specific RECOGNIN, ASTROCYTIN MALIGNIN,RECOGNIN L, RECOGNIN M, or their CHEMORECIPROCALS of the normal tissueor component which they will neighbor and thus being "recognized" as"foreign" and thus avoid the reactions to alien substances, includingrejection.

Another aspect of this invention is the production of a valuablespecific antibody-like product (Anti-Astrocytin) to a specific brainproduct (Astrocytin), permitting the use of this antibody-like productto specifically complex with and, as a specific delivery vehicle to,specific points in the nervous system of all species. MALIGNIN,ASTROCYTIN, RECOGNIN M and RECOGNIN L are RECOGNINS.

Still another aspect of this invention is the production from biologicalfluids of two new products, TARGET-ATTACHING-GLOBULINS (TAG), which areso named because they are produced by two reactions, the first reactingbiological fluids with a synthetic complex containing physiochemicalconfigurations which mimic those of the MALIGNIN, RECOGNIN L andRECOGNIN M and called TARGET, the second, cleaving the specific TAG fromthe complex, and by the measure of the TAG so produced obtaining aquantitative indication from the biological fluids of living organismswhether there is present a tumor in that organism; hence a diagnostictest for tumors. Because TAG products and ANTI-MALIGNIN arephysicochemically complimentary to MALIGNIN, they are termedCHEMORECIPROCALS.

It has been further discovered that two quantitatively and qualitativelydistinct TAG products can be produced depending upon the time permittedfor the reaction of serum with the specific TARGET reagent used, anddepending upon the time permitted for the cleavage of the product whichhas been complexed.

After examining the amounts of these products which could be producedfrom a number of different individuals with brain tumors and variousother medical disorders, as well as in those with no apparent diseaseprocess, it became apparent that the amounts of those two new productswhich could be produced in a given individual was indicative of whetherthat individual had a brain tumor, hence a novel serum diagnostic testfor brain tumors has been discovered.

The utility of these new products, in addition to their use to diagnosefrom serum and other biological fluids the presence of brain and othertumors, is illustrated by the demonstration that TAG and anti-RECOGNINcompounds attach to glial tumor cells preferentially in histologicalsection of brain tumor and surrounding tissue removed at surgery of thebrain tumor. This preferential labelling by TAG and Anti-RECOGNINS oftumor cells is demonstrated through standard immunofluorescenttechniques. Thus a new method is also available for determining throughhistological examination with a new degree of certainty whether tumorcells have penetrated to the very edges of the tissues removedindicating the likelihood that tumor still remains in the brain or otherorgan, or that tumor cells are absent from the periphery of the tissueremoved, indicating the possibility that all of tumor has been removedfrom the brain or other organ. In addition, TAG and Anti-RECOGNINSproduced as described have been found to be cytotoxic for glioma braintumor cells grown in tissue culture in vitro. This high affinity fortumor cells in another medium, here grown in tissue culture, is furtherevidence of the specific-coupling potential of the new products TAG, andexplains the adoption of the name TARGET-ATTACHING-GOBULINS (TAG) as doTAG's properties in regard to the synthetic product TARGET, and to tumorcells in histological section. Further, the cytotoxicity of TAG andanti-RECOGNINS for tumor cells provides an additional new diagnostictest for serum of patients who are suspected of suffering from a tumor.Thus, for example, the serum or other body fluid of these patients isreacted with TARGET to produce TAG and the product TAG is tested intissue culture growths of tumor cells for cytoxicity. Both theconcentration of TAG and the degree of cytoxocity manifested by the TAGwhich can be produced from a given individual's serum may be not onlydiagnostic but also of value in tracing the course of the disorderpreoperatively and postoperatively in a given patient. Coupling ofradioactive and dye tracers to TAG provides new TAG products which areuseful in vivo in the diagnoses of tumors and in their exactlocalization. Thus the injection of suitably labelled TAG eitherintra-arterially or intravenously, into the cerebrospinal fluid, ordirectly into brain tissue or its cavities, permits the demonstration byradioactive means, or by visualization of the coupled dye, of thepresence of a brain tumor, for it is only to the tumor cells that theTAG specifically attaches. Further, this method permits the precisevisualization of the location of the brain tumor. This can be seen to bean improvement of this in vivo diagnostic method using anti-ASTROCYTINproduced in rabbit blood to label the brain tumor, because the use ofTAG produced from human serum avoids the possibility of foreign proteinreactions. Since TAG and anti-RECOGNIS have the chemical specificitywhich permits preferential attachement to ASTROCYTIN precursorcontaining tumor cells both in vitro and in vivo, these products mayalso be used therapeutically, as well as diagnostically, when coupled,e.g., with radioactive, proton capture agents, or other toxic physicalor chemical agents, so that these toxic substances may be localizedpreferentially through these compounds' specificity of attachement inthe tumor cells as compared to their neighboring normal cells. Thisselectivity is universally recognized as the crucial, or at least onecrucial factor for achieving effective chemical or physical therapy oftumors, and a factor which has hitherto not been achieved. Thus TAG hasdemonstrated efficacy in attaching preferentially to the tumor cells,and should have promise as a new therapeutic product for these reasons.

In the serum of patients with malignant tumors, as will be seen in theexamples below, one type of TAG, SLOW-TAG (S-TAG) as distinguished fromFAST-TAG (F-TAG), can be produced in relatively greater amonts from agiven volume of serum than in patients without such tumors. Thissuggests that either one of TAG'S naturally occuring precursors (P-TAG)is increased in concentration or that other factors exist which favorthe relative in vitro production of S-TAG over F-TAG.

The possible relationship of the function of the actual syntheticproducts TARGET and TAG to their precursors, and in turn to thefunctions of postulated but not demonstrated cell "antigens" andcirculating "antibodies" to them which may exist in vivo has yet to beelucidated. Thus for example, in antibody-like fashion, F-TAG and S-TAGproduce single discrete lines of reaction with ASTROCYTI in Ouchterlonygel diffusion, and the injection of TARGET in rabbits induces anincrease in the yield of TAG products from rabbit serum after reactingwith TARGET. The finding that there may be a normal level of a precursorresembling antibody to a cell antigen which is hidden in thenon-dividing cell raises a question as to the possible function of thepair. It is here proposed that TAG precursor (P-TAG) and TARGET-likesubstances exist in vivo which function in the control of cellproliferation and cell death. Thus, for example, the exposure of a cellconstituent which normally is not directly exposed to serum proteins mayoccur during cell division. The exposure of this cell constituent couldresult in that constituent becoming converted to a TARGET-like substanceto which the attachment of a P-TAG-like molecule from serum may thenoccur, which would stimulate cell division or inhibit it. Alternatively,a non-dividing cell which is injured or malfunctioning may expose aTARGET- like substance to which the attachement of P-TAG-like moleculesmay be reparative. However, under certain cell conditions theattachement of P-TAG-like molecules may induce the destruction of thecell (e.g., ANTI-GLIOMA-TAG synthetically produced as here described ismarkedly cytotoxic to glioma tumor cells growing in tissue culture).This could thus represent a mirror of a normal mechanism for the controlof cell division, and for either the repair or the removal of individualcells in the body throughout the life of the organism. If the exposureof cell constituents is abnormally increased to that abnormally largeamounts of cell TARGET-like substances are formed, as may occur inrapidly dividing cancer cells such as in brain gliomas, an increase inthe concentration of one type of serum P-TAG relative to another may beinduced.

Whatever the actual function of the precursors, the increase in therelative amount of predominately one type of TAG, SLOW-TAG (S-TAG) whichcan be produced in vitro by the methods here described from the serum ofpatients with malignant tumors is the basis of the serum diagnostic testdescribed in the examples which follow.

EXAMPLE I Production of Crude ASTROCYTIN-Precursor-Containing Fraction

Human brain glioma tumor tissue, removed at surgery, is dissected freeas possible of surface blood vessels and normal brain tissue. For atypical amount of dissected tumor tissue of 11 grams, the tissue isweighed into six 1.5 g. and two 1.0 g. aliquots. Each aliquot is thentreated as follows.

Each aliquot is homogenized in neutral buffer solution by sonificationor other mechanical means. For example, each aliquot is homogenized in100 cc per g. of tissue of 0.005M phosphate buffer solution, pH 7, in aWaring blender. Homogenization should be done in the cold to preventdenaturation of proteins. For example, the blender should be precooledin a cold room at 0°-5° C. and operated for about only three minutes.

The homogenate is then centrifuged for clarification, for example at80,000 times gravity for 30 minutes in a refrigerated ultracentrifuge.The soluble supernatant is decanted and kept in the cold. The insolubleresidue is rehomogenized with a further 100 cc of neural buffer andcentrifuged as before, and the second soluble extract combined with thefirst. Best yields are obtained when this procedure of homogenizationand centrifugation is repeated until less than 50 micrograms of proteinper ml. of solution are obtained in the supernate. With most tissue thisis accomplished by the fifth extraction.

The solutions thus obtained are combined and concentrated byperevaporation with subsequent dialysis, as by dialysis against 0.006Mphosphate buffer in the cold to produce a volume of 15 ml. The volume ofthis solution is noted, an aliquot is taken for total protein analysis,and the remainder is fractionated to obtain the protein fraction havinga pK range between 1 and 4. The preferred method of fractionation ischromatography as follows.

The solution is fractionated in the cold room (4° C.) on a DEAEcellulose (Cellex-D) column 2.5×11.0 cm., which has been equilibratedwith 0.005M sodium phosphate buffer. Stepwise eluting solvent changesare made with the following solvents (solutions): Solution (1) 4.04 g.NaH₂ PO₄ and 6.50 are dissolved in 15 liters of distilled H₂ O (0.005molar, pH 7); Solution (2) 8.57 g. NaH₂ PO₄, is dissolved in 2480 ml. ofdistilled H₂ O; Solution (3) 17.1 g. of NaH₂ PO₄ is dissolved in 2480ml. of distilled H₂ O; (0.05 molar, pH 4.7); Solution (4) 59.65 g. ofNaH₂ PO₄ is dissolved in 2470 ml. distilled H₂ O (0.175 molar); Solution(5) 101.6 g. of NaH₂ PO₄ is dissolved in 2455 ml. distilled H₂ O (0.3molar, pH 4.3); Solution (6) 340.1 g. of NaH₂ PO₄ is dissolved in 2465ml. of distilled H₂ O (1.0 molar, pH 4.1); Solution (7) 283.64 g. of 80%phosphoric acid (H₃ PO₄) is made up in 2460 ml. of distilled H₂ O (1.0molar, pH 1.0).

Add nervous tissue extract, 6 to 10 ml. volume. Let it pass into column.Then overlay with Solution (1) and attach a reservoir of 300 ml. ofSolution (1) to drip by gravity onto the column. Three ml. aliquots ofeffluent are collected by means of an automatic fraction collector. Thesubsequent eluting solutions are exchanged stepwise at the followingelution tube numbers. Solution (2): at tube 88, bring solution on columnto top of resin, then overlay and attach reservoir of 50 ml. of Solution(2); Solution (3): at tube 98, bring solution on column to top of resin,then overlay and attach reservoir of 75 ml. of Solution (3); Solution(4): at tube 114, bring solution on column to top of resin, then overlayand attach reservoir of 150 ml. of Solution (4); Solution (5): at tube155, bring solution on column to top of resin, then overlay and attachreservoir of 125 ml. of Solution (5); Solution (6): at tube 187, bringsolution on column to top of resin, then overlay and attach reservoir of175 ml. of Solution (7); continue eluting until at tube 260, elution iscomplete. Use freshly prepared resin for every new volume of tissueextract. Each effluent tube is quantitatively analyzed for protein. Theelutes in the tube numbers 212 to 230 are combined, and contain thecrude products from which ASTROCYTIN will be produced.

While data has been published on this crude material, called fraction10B in the past, [Protein Metabolism of the Nervous System, pp 555-69(Pleum Press, 1970); Journal of Neurosurgery, Vol. 33, pp 281-286(September, 1970)] the cleavage from fraction 10B of the specificproduct herein called ASTROCYTIN has now been accomplished. Crudefraction 10B can be prepared as a product in amounts between 0.1 and 10mg. per gm. of original fresh nervous system tissue from which it wasobtained. In addition to an ASTROCYTIN-precursor it contains varyingamounts of covalently bound carbohydrate residues including a number ofhexoses, namely glucose, galactose, mannose; hexosamines, includingglucosamine, galactosamine and mannosamine; and occasionally othersugars, such as fructose, ribose and perhaps rhamnose. It also containslarge molecular weight protein products, several lipids and nucleicacids.

EXAMPLE 2 Production of Purified ASTROCYTIN from CrudeASTROCYTIN-Precursor-Containing Fraction

The ASTROCYTIN-Precursor-Containing fraction is further isolated fromcontaminants. In the preferred embodiments, the material from Example 1is chromatographed on Sephadex G-50 resin with a typical column of 40cm. long, 2.5 cm. diameter, and 196 ml. volume. The pressure used is 40mm. Hg.,; the flow rate is 35 ml. per hour, and the buffer is 0.05 molarphosphate buffer solution, pH 7.2. The first (flow through) peakcontains ASTROCYTIN-Precursor together with impurities, whereassubsequent peaks contain only impurities.

In the preferred embodiment, the products in the above firstflow-through peak are then concentrated on Sephadex G-15, then passedonto a column of Cellex-D with the same solutions (1) through (7) asExample 1, and the same elution steps as performed in Example 1. Theproducts ASTROCYTIN is present as a sharp peak in the same tubes (number212-230) as before, thus maintaining its behaviour on Cellex-Dchromatography without the presence of a large number of contaminants.

Low molecular weight contaminants may then be removed by techniquesknown to the art, such as millipore disc filtration. In the preferredmethod, the product ASTROCYTIN is freed of salt and other smallmolecular weight contaminants by filtration through Millipore PelliconDisc No. 1000, 13 mm., which retains substances of molecular weightsgreater than 1000 and permits to pass through those of molecular weightless than 1000. The product ASTROCYTIN remains on the Pellicon Disc, andis recovered from it by washing with Solution (1) of Example 1.

ASTROCYTIN is then obtained by isolating the compound having a molecularweight of about 8000 from the above solution. A preferred method usesthin layer gel (TLG) chromatograph as follows:

The apparatus used is the commercially available one designed byBoehringer Mannheim GmbH; Pharmacia Fine Chemicals and CAMAG(Switzerland). The resin 2.5 g. of Sephadex G-200 superfine is preparedin 85 ml. of 0.5M. NaCl in 0.02M. Na₂ HPO₄ KH₂ PO₄ Phosphate Buffer ph6.8 (6.6-7.0). Allow to swell two or three days at room temperature withoccasional gentle mixing. (Magnetic and other stirrers should not beused). The swollen gel is stabilized for three weeks at refrigeratortemperature; however, bacterial and fungal growth may interfere with theswollen gel. If the gel is to be kept for longer periods of time, asmall amount of a bacteriostatic agent should be added (sodium Azide0.02%). 2.5 g. of dry gel are used to make two 20×20 cm. glass plates of0.5 mm. thick. The plates are either allowed to dry at room temperaturefor 10 minutes and transferred to a moist chamber where they can bestored for about two weeks, or they are used immediately afterappropriate pre-equilibration. (Usually during the night for a minimumof 12 hours). The main function of equilibration is to normalize theratio between the stationary and mobile phase volumes. With thepre-equilibrated plates in a horizontal position, substances to bedetermined are applied with micropipettes as spots or as a streak at thestart line. 10 ml. to 20 ml. of 0.2-2% of protein solution is placed onthe edge of a microscopic cover slide (18 by 18 mm.) and held againstthe gel surface. In a few seconds the solution will soak into the gel.All samples are first prepared on the cover slides and then quicklyapplied. If not enough material is used, it is difficult to locateindividual spots after separation. If too much material is applied nodefined separation occurs. The samples are diluted with buffer foreasier handling and the separation of samples is carried in a descendingtechnique with the plate at an angle of 22°. The flow rate of about 1-2cm/hour is most suitable. Marker substances (such as cytochrome C,hemoglobin, myoglobin or bromophenol blue labeled albumin) are appliedat different positions across the plate and also to serve as referenceproteins for calculation of relative distance (mobility) of unknowns.After application of samples, the plates are replaced in the apparatusand the paper wick pushed slightly downwards to ensure good contact withthe gel layer. The paper wick must not drip. Excess moisture is wipedoff. The liquid solvent in the reservoir is kept constant at 1 cm. fromthe upper end of the vessel. The runs are usually completed in 4 to 7hours depending on the progress of separation. With colored substancesseparation follows directly. The separated spots of protein are easilymade visible by transferring them to a paper sheet replica of TLG plateafter the chromatographic separation has been completed, and by stainingthem on the prewashed methanol +H₂ O+ acetic acid--90:5:5, for 48 hours.The paper sheet is 3 mm. filter paper. A sheet of paper 20×18 cm. isplaced over the gel layer and pressed (rolled) just enough to ensurecontact with the gel. Care is taken not to trap air under the paper(replica) and not to disturb the gel layer. The liquid phase is soakedoff from the gel layer by the paper and removed after about one minute,immediately dried in an oven at 60° temperature for 15 minutes andstained in the normal way with any of the routine staining procedures.Staining is performed by spraying the replica-paper with 0.03%diazotized sulfanilic acid in 10% Sodium Carbonate (Pauley's Reagent).Staining can also be accomplished with a saturated solution of AmidoBlack in Methanol-Acetic Acid (90:10v/v is used); the staining time is5-10 minutes. For destaining, rinse with two volumes of the 90:10methanol and acetic acid solution mixed with one volume of H₂ O. It isdifficult to obtain low background staining without very extensivewashing. The plates themselves may also be dried at about 60° C. (in anoven with air circulation) but only if the ASTROCYTIN is to be stained.For isolation purposes, the plate should only be air dried at roomtemperature. Over-heating can led to cracking, but this can usually beavoided with at 50°-60° C. temperature which dries a sephadex G-200plate in 15-30 minutes. The dry plates are allowed to swell for 10minutes in a mixture of methanol +H₂ O+ acetic acid (75:20:5) andstained in a saturated Amido Black in the same solvent system for fivehours and subsequently washed by bathing for two hours in the samesolvent before they are dried. For molecular weight determinations thedistance from the starting line to the middle of each zone is measuredwith an accuracy of 0.05 mm. either directly on the print (replica) oron the densitogram. The result is expressed by the R_(m) value definedas the ratio of the migration distance of the tested protein (d_(p)) tothat of cytochrome C or myoglobin (d_(m)) which is used as the referenceprotein: Relating migration distance of tested substance to standard isthe formula (-R_(m) =d_(p) /d_(m)). A straight calibration line isobtained by plotting the logarithm of the molecular weight of thestandards used against the R_(m). From this line the molecular weight ofthe unknown protein can be obtained. For most exact results mix equalparts of the protein sample solution with standard, in this case,Cytochrome C, before applying to the plate. By the above TLG procedurethe produce ASTROCYTIN in observed as a discrete spot at a distance ofapproximately 0.83±0.02 with reference to the standard Cytochrome C,yielding an approximate molecular weight of 8000 for ASTROCYTIN. Severaldiscrete products are separated in this procedure from ASTROCYTIN on thebasis of slight differences in molecular weight. Thus, three productscarried as contamination to this point with molecular weight ofapproximately 64,000, 148,000 and 230,000, and one occasionally ofmolecular weight 32,000, have been detected and removed by the TLGmethods described above. The product ASTROCYTIN is aspirated with thegel in which it is contained, in dry form, dissolved in Solution (1) andfreed of resin by centrifugation or other similar means.

The product ASTROCYTIN which has been produced at this stage is solublein distilled water, soluble at neutral and acid pH, and insoluble atalkaline pH and has a spectrophotometric absorption peak wavelength of280 mu. It is a polypeptide with molecular weight, as stated above, ofapproximately 8000. Its covalently linked amino acids are shown byhydrolysis with 6N HCl then quantitative automatic determination to havethe following average composition of amino acids:

    ______________________________________                                                      Approximate Number                                                            of residues                                                     ______________________________________                                        Aspartic acid   9                                                             Threonine       5                                                             Serine          6                                                             Glutamic acid   13                                                            Proline         4                                                             Glycine         6                                                             Alanine         9                                                             Valine          4                                                             1/2 Cysteine    2                                                             Methionine      1                                                             Isoleucine      2                                                             Leucine         8                                                             Tyrosine        2                                                             Phenylalanine   3                                                             Lysine          8                                                             Histidine       2                                                             Arginine        4                                                             Approximate Total                                                                             88                                                            ______________________________________                                    

Cysteic acid, hydroxyproline, norleucine, ammonia, isodesmosine,desmosine, hydroxyysine, lysinonorleucine and gamma-aminobutyric acidare all absent indetectable amounts, but a trace of glucosamine may bepresent.

From 11 grams of the starting brain tumor tissue in EXAMPLE 1,approximately 3 mg. of purified ASTROCYTIN is produced by the abovemethods.

EXAMPLE 2A PRODUCTION OF "REELER" RECOGNIN

Reeler disease is a genetic disorder in which animals fail to achievestable coordinated motor activity, producing a reeling state, due to thefailure of migration of certain nerve cells to a particular place in thebrain, the cerebellum, at a particular developmental time. Particularglial cells have been shown, in electron microscopic studies, to providethe vertical pole-like axes along which the nerve cells migrate to theirnew positions in the normal state. The failure of nerve cells to climbthese glial fibers, in Reeler disease is thought to be due to somedisturbance in the nerve cells or the glia or both.

Following the methods of Examples 1 and 2, Recognin from mouse reelerbrain was produced and compared with Recognin produced from normal mousebrain. The molecular weight of the Recognin produced from all areas ofnormal mouse brain was 8,000. The molecular weight of "Reeler" Recogninwas 3,600 to 5,000. "Reeler" Recognin is abnormal, as shown by its muchsmaller molecular weight. Furthermore, the amount of Recognin which canbe produced rom the cerebellum of reeler mouse brain is much reducedwhen compared to the normal. This is shown in Table I.

                  TABLE I                                                         ______________________________________                                        Recognin Concentration in Mouse Brain, mg/g.                                                     Normal Reeler                                              ______________________________________                                        Cerebellum (16-day old mouse)                                                                      2.50     0.71                                            Cortex (16-day old mouse)                                                                          0.60     0.96                                            Cortex (4-day old mouse)                                                                           0.29     0.53                                            Brainstem (16-day old mouse)                                                                       0.90     1.64                                            Whole Brain (17-day old mouse)                                                                     1.27     1.53                                            Whole Brain (1-day old mouse)                                                                      5.00     5.86                                            ______________________________________                                    

Table I shows that while there is a marked decrease in the concentrationof Recognin in cerebellum of Reeler mouse, there are the same or greateramounts of Recognins in other areas of the brain. The Recognin may notmove from the other areas of brain to the cerebellum, or the slightincrease in other areas of Reeler brain may reflect some compensatoryaction.

This pathological Recognin in Reeler brain is correlated with theinability for migrating brain cells to make the contacts they must inorder to achieve proper placement, confirming the role of RECOGNINS inrecognition and learning in cells.

By analogy the ANTI-RECOGNINS to Recognin produced from mouse reelerbrain may be produced and used in mice in a manner similar to the usesof ANTI-ASTROCYTIN and ANTI-MALIGNIN, as described herein.

EXAMPLE 3 Production of MALIGNIN-Precursor in Artifical Cancer CellCulture Fermentations

Generally, sterile technique is scrupulously maintained.

All solutions (e.g. Hank's Balanced Salt (BSS), F-10 Nutrient medium,fetal calf serum, trypsin solution) are incubated at about 35° C. in awater bath for approximately 20 minutes or more before use.

Cells are removed from tumor tissue and grown in vitro for manygenerations using a suitable medium, such as described below. Pre-rinsebeakers to be used with a sterilizing solution, for example 12-proponalplus amphyl or creolin solution.

In the preferred embodiment, the artificial cancer cells (i.e., cellsgrown in vitro for many generations) are grown in 250 ml. flasks. Theliquid medium in which the cells are growing is discharged into thepre-rinsed beakers. The cells are growing is discharged into thepre-rinsed beakers. The cells are then washed gently with 5-10 ml. ofHank's BSS or other similar solution for about 30 seconds. Avoidagitation. All walls and surfaces are washed. The solution is clarifiedof cells by centrifugation in the cold from minutes at 3,000 rpm. Themedium is poured into a beaker as above. Add a small amount of bufferedproteinase enzyme solution and rinse quickly to avoid digestion of thecells. In the preferred method, 1-2 ml. of trypsin solution (EDTA) isadded and rinsed for only 10 seconds. Pour off the trypsin solution.

Add a similar volume of fresh trypsin solution and incubate until thecells are seen to be separated from the walls of the chamber throughmicroscopic observation. This usually requires 5-10 minutes. Add asuitable growth medium, such as 50 ml. of a solution of 7-10 percentsolution of fetal calf serum in 100 ml. of F-10 Nutrient medium.

Twenty five ml. of the fresh medium with cells is transferred to a newgrowth chamber for propagation. Both chambers are placed in an incubatorat 35° C. for approximately seven days. By the procedure of this Exampleto this point, an artificial cancer cell culture is divided into twofresh cultures approximately every seven days. This entire procedure maybe repeated as often as desired, at approximately seven-day intervals,for each growth chamber. Thus, the number of cells growing in vitro maybe doubled approximately every seven days.

The cells may be extracted for the production of MALIGNIN afterapproximately seven days of growth. For example, cells growing in each250 ml. growth chamber as described above, may be recovered as follows.

The medium is transferred to a centrifuge tube and centrifuged at 3,000rpm in the cold for 10 minutes. The medium is discarded. The cellsremaining in the growth chamber are scraped from the chamber walls andwashed into the centrifuge tubes with neutral buffer solution. The cellsare washed twice with neutral buffer solution, centrifuged again at3,000 rpm in the cold, and the medium is discarded. The washed cells aresuspended in 10 ml. of neutral phosphate buffer until ready forextraction of crude MALAGNIN-Precursor-Containing fraction.

EXAMPLE 4 Production of Crude MALIGNIN-Precursor-Containing Fraction

Washed cells suspended in neutral buffer from Example 3 are mechanicallydisrupted under conditions which avoid denaturation of most proteins. Inthe preferred method, the washed cells are treated in the cold with asonifier for 20 seconds.

After sonification the cell residues are centrifused at 30,000 rpm for30 minutes and the supernatant decanted. Ten ml. aliquots of buffersolution are used to wash remaining cell residues. Sonify and centrifugeas above and combine the supernatants. Repeat the process once more.

The combined supernant is perevaporated to reduce the approximate 30 ml.volume to about 6-7 ml. An aliquot is taken for total protein analysisand the remainder is fractionated according to the methods of EXAMPLE 1for ASTROCYTI Precursor.

EXAMPLE 5 Production of Purified MALIGNIN Product from CrudeMALIGNIN-containing Fraction

The product MALIGNIN is further isolated from contaminants by themethods of EXAMPLE 2 for ASTROCYTIN.

In the TLG step of the preferred embodiment, the product MALIGNIN isobserved as a discrete spot at a distance of approximately 0.91±0.02with reference to the standard cytochrome C, yielding an approximatemolecular weight of 10,000 for MALIGNIN.

The product MALIGNIN which has been produced at this stage is soluble indistilled water, soluble at neutral or acid pH, and insoluble atalkaline pH and having a spectrophotometric absorption peak of 280 mu.It is a polypeptide with molecular weight of approximately 10,000.

The molecular weights of MALIGNIN produced in fermentation culturesstabilized in successive generations of the cultures as shown by thethin layer gel chromatography determinations are set forth in Table II.The reproductivity of the molecular weight determinations is remarkablein view of the inherent limitations of TLG chromatography.

                  TABLE II                                                        ______________________________________                                        Reproducability of Molecular weight of Malignin produced.                     Run No.                                                                              Mol. Wt. Run No.  Mol. Wt.                                                                              Run No.                                                                              Mol. Wt.                              ______________________________________                                        1       9,500    9       10,100  17     10,180                                2       8,900   10       10,180  18     10,190                                3      10,000   11       10,180  19     10,190                                4      10,050   12       10,180  20     10,180                                5      10,100   13       10,180  21     10,000                                6      10,000   14       10,050  22      9,500                                7      10,150   15       10,180  23     10,180                                8      12,500   16       10,190                                               ______________________________________                                    

MALIGNIN'S covalently linked amino acids are shown by hydrolysis with 6NHCl then quantitative determination to have the following averagecomposition of amino acids:

    ______________________________________                                                      Approximate Number                                                            of Residues                                                     ______________________________________                                        Aspartic acid   9                                                             Threonine       5                                                             Serine          5                                                             Glutamic acid   13                                                            Proline         4                                                             Glycine         6                                                             Alanine         9                                                             Valine          6                                                             1/2 Cysteine    1                                                             Methionine      2                                                             Isoleucine      4                                                             Leucine         8                                                             Tyrosine        3                                                             Phenylalanine   3                                                             Lysine          6                                                             Histidine       2                                                             Arginine        5                                                             Approxiamate Total                                                                            89                                                            ______________________________________                                    

The amino acids cysteic acid, hydroxyproline, norleucine, ammonia,isodesmosine, desmosine, hydroxylysine, lysinonorleucine andgamma-aminobutyric acid being absent in detectable amounts.

A typical yield of pure MALIGNIN from twelve 250 ml. reaction chambersof EXAMPLE 3 together is approximately 1 mg. of MALIGNIN.

EXAMPLE 5A Production of RECOGNIN L

Malignant cells grown in tissue culture, a lymphocytic lymphoma line ofcells designated P₃ G, were obtained from Mason Research Institute,Rockville, Md.

Approximately 1 gm of packed cells of each of these were not furtherpropagated upon receipt but extracted immediately according to theidentical protocol used for producing Malignin from glioma cells(EXAMPLES 3, 4 and 5). Thus, the entire medium plus cells wastransferred to centrifuge tubes with cold 0.005M phosphate buffer, pH 7,and centrifuged at 3,000 rpm in the cold for 10 minutes, the mediumdiscarded, the cells washed twice with cold buffer, centrifuged againtwice as before, and the washings discarded. The washed cells weresuspended in the same buffer and disrupted by sonification for 20seconds. The cell residues were centrifuged at 30,000 rpm for 30minutes, the solubilized protein in the supernatant decanted andcollected, and the cell residues sonified twice more, until no furtherappreciable protein was solubilized. The solubilized protein wasconcentrated and the recognin cloven and purified by Cellex D (BioRad)and Sephadex 200 (Pharmacia) gel chromatography. The yield, molecularweight, amino acid composition, behavior on thin layer gelchromatography and immunological properties of this polypeptide similarto those of Astrocytin and Maligin. The yield in the case of P₃ G cellswas approximately 1 mg/g wet weight of cells.

The covalently linked amino acids of Recognin L are shown, by hydrolysis(in vacuo) with 6N HCL at 108° C. for 12 hours followed by quantitativedetermination, to have the following average composition of amino acids(the nearest integer for the mole number of each amino acid is theaverage of two separate determinations):

    ______________________________________                                                     Number of Residues                                               ______________________________________                                        Threonine      5                                                              Serine         5                                                              1/2 Cysteine   1                                                              Methionine     1                                                              Valine         6                                                              Isoleucine     4                                                              Phenylalanine  3                                                              Lysine         6                                                              Histidine      2                                                              Arginine       5                                                              Aspartic Acid  8                                                              Glutamic Acid  10                                                             Leucine        7                                                              Tryosine       1                                                              Proline        5                                                              Glycine        13                                                             Alanine        10                                                                            92                                                             ______________________________________                                    

EXAMPLE 5B Production of RECOGNIN M

Malignant cells grown in tissue culture, a mammary carcinoma cell linedesignated MCF-7, were obtained from Mason Research Institute,Rockville, Md.

Approximately 1 gm of packed cells of MCF-7 was not further propagatedupon receipt but extracted immediately according to the protocol usedfor producing Malignin from glioma cells. (EXAMPLES 3, 4 and 5). Thus,the entire medium plus cells was transferred to centrifuge tubes withcold 0.005M phosphate buffer, pH 7, and centrifuged at 3,000 rpm in thecold for 10 minutes, the medium discarded, the cells washed twice withcold buffer, centrifuged again twice as before, and the washingsdiscarded. The washed cells were suspended in the same buffer anddisrupted by sonification for 20 seconds. The cell residues werecentrifuged at 30,000 rpm for 30 minutes, the solubilized protein in thesupernatant decanted and collected, and the cell residues sonified twicemore, until no further appreciable protein was solubilized. Thesolubilized protein was concentrated and the recognin cloben andpurified by Cellex D (BioRad) and Sephadex 200 (Pharmacia) gelchromatography. The yield, molecular weight, amino acid composition,behavior on thin layer gel chromatography and immunological propertiesof this polypeptide is similar to those of Astrocytin and Maligin, theprevious two cancer recognins described. The yield in the case of MCF-7cells was approximately 1 mg/g wet weight of cells.

The covalently linked amino acids of Recognin M are shown, by hydrolysis(in vacuo) with 6N HCL at 108° C. for 12 hours followed by quantitativeautomatic determination, to have the following average composition ofamino acids (the nearest integer for the mole number of each amino acidis the average of two separate determinations):

    ______________________________________                                                     Number of Residues                                               ______________________________________                                        Threonine      5                                                              Serine         5                                                              1/2 Cysteine   1                                                              Methionine     1                                                              Valine         6                                                              Isoleucine     4                                                              Phenylalanine  3                                                              Lysine         6                                                              Histidine      2                                                              Arginine       5                                                              Aspartic Acid  9                                                              Glutamic Acid  11                                                             Leucine        8                                                              Tryosine       2                                                              Proline        4                                                              Glycine        9                                                              Alanine        9                                                                             90                                                             ______________________________________                                    

The unique structures of MALIGNIN, ASTROCYTIN, RECOGNIN M and RECOGNIN Lwere confirmed by an exhaustive computerized search which compared theircompositions with virtually all known protein substances.

The amino acid composition of MALIGNIN and ASTROCYTIN, their absoluteand relative amounts of each amino acid component in terms of the totalnumber of amino acid residues per mole, and their absolute and relativeamounts of each amino acid component in terms of the molecular weight ofthe molecule, were submitted to matrix computer analysis against thelargest known library of protein structure in the world, that of theNational Biomedical Research Foundation, Washington, D.C. No structureidentical to or even very close to that of ASTROCYTI, MALIGNIN, RECOGNINM or RECOGNIN L was found in the matrix analysis comparison with severalhundred thousand proteins and protein fragments.

The only proteins that were structurally related in any way are shown inTable III with their individual amino compositions and molecular weightsfor comparison. The computer is programmed to identify, from the severalhundred thousand possibilities, any degree of similarity in structure.Thus, for example, proteins of molecular weight much larger or smallerwill not match, nor those with less than 85 or greater than 95 residues,nor those with less than 10 or greater than 15 glutamic acid residues,nor those with less than 6 or greater than 11 aspartic residues, and soon for each of the twenty amino acids involved.

                                      TABLE III                                   __________________________________________________________________________    COMPARISON OF THE STRUCTURES OF RECOGNINS                                     TO NEAREST STRUCTURES BY COMPUTER SEARCH                                                                  Cyto- Ferre-                                                                              Ferre-                                                                             Acyl-                                                                             Neuro-                                                                             Neuro-                                                                            Gonad-                     Astro-     Recognin                                                                           Recognin                                                                           chrome                                                                              doxin doxin                                                                              Carrier                                                                           physin                                                                             physin                                                                            otropin                    cytin                                                                              Malignin                                                                            M    L    b.sub.5                                                                             Luc. G1                                                                             Alf. E. Coli                                                                           Bovine                                                                             Pig Releas.             __________________________________________________________________________    Aspartic                                                                             9    9     9    8    9     10    8    7   2    3   0                   acid                                                                          Threonine                                                                            5    5     5    5    6     4     6    6   2    2   0                   Serine 6    5     5    5    5     7     8    3   6    7   1                   Glutamic                                                                             13   13    11   10   14    13    13   14  9    9   0                   acid                                                                          Proline                                                                              4    4     4    5    3     5     3    1   8    7   1                   Glycine                                                                              6    6     9    13   6     7     7    4   16   14  2                   Alanine                                                                              9    7     9    10   4     6     9    7   6    7   0                   Valine 4    6     6    6    4     6     9    7   4    2   0                   1/2 Cysteine                                                                         2    1     1    1    0     5     5    0   14   14  0                   Methionine                                                                           1    2     1    1    1     0     0    1   1    1   0                   Isoleucine                                                                           2    4     4    4    4     4     4    7   2    2   0                   Leucine                                                                              8    8     8    7    7     10    6    5   6    7   1                   Tyrosine                                                                             2    3     2    1    3     3     4    1   1    1   1                   Phenylalanine                                                                        3    3     3    3    3     3     2    2   3    3   0                   Lysine 8    6     6    6    7     5     5    4   2    2   0                   Histidine                                                                            2    2     2    2    7     1     2    1   0    0   1                   Arginine                                                                             4    5     5    5    3     2     1    1   7    5   1                   Asparagine                                                                           0    0     0    0    0     0     1    2   3    2   0                   Tryptophane                                                                          0    0     0    0    1     1     1    0   0    0   1                   Glutamine                                                                            0    0     0    0    0     4     3    4   5    4   0                   Total No.                                                                            88   89    90   92   87    96    97   77  97   92  10                  Residues                                                                      Molecular                                                                            8,000                                                                              10,000                                                                              8,000                                                                              8,000                                                                              10,035                                                                              10,493                                                                              8,509                                 Weight                                                                        __________________________________________________________________________

This `fingerprint` thus has some 22 individual variables to match. Somesubstances will match on one, on four or on five variables but nonematch on all 22. In fact, none match in better than 14 variables leavingdifferences in 8 variables.

Thus, the closest fit is cytochrome b₅ (human). As seen in Table III,the alanine, argine, asparagine, aspartic acid, cysteine, glutamine,histidine, methionine and tyrosine and tryptophane residue numbers alldiffer appreciably to markedly from the Recognins. Because cytochrome b₅contains 7 histidines while Astrocytin, Malignin, Recognin L andRecognin M contain only 2, they could not possibly have the samechemical structure.

The most unusual thing about the composition of Astrocytin, Malignin,Recognin L and Recognin M is the high concentration of glutamic acid.One would expect to find only 5 or 6 residues in 89.

Other next-closest fits are the ferrodoxins of leucaene glauca and ofalfalfa, respectively, but these also differ markedly in four and sixamino acids, respectively, and appreciably in two others, and in having96 and 97 residues, respectively. The next-closest fit is theacylcarrier protein of E. Coli 26, but this also differs markedly ineleven amino acids from Malignin, Astrocytin, Recognin L and Recognin Mand has only 77 residues.

Some other brain proteins (neurophysin, bovine and pig, and gonadotropinreleasing hormone) are listed in Table III to illustrate how much worsethe match is for the remaining several hundred thousand proteinfragments in the computer memory bank.

Respiratory proteins may contain metals and/or heme components in theirin situ state, but the isolated protein fragment, e.g., forapocytochrome b₅ contains neither. Additional microanalysis ofAstrocytin and Malignin has shown them to be free of iron, sulfur,phosphorus and magnesium (all less than 0.01%), and the spectralcharacteristics show typical absorption at 280 mu. Upon recombination ofheme with apoprotein, the typical absorption spectra between 400 and 450m are restored.

Despite the structural uniqueness of Astrocytin, Malignin, Recognin Land Recognin M from all other proteins and protein fragments, it isnoteworthy and perhaps of great importance, that the closest structuresare those of respiratory proteins. It is well known to the art that manyimportant relationships can be drawn from both a developmental geneticpoint of view and from a functional point of view in the type ofstructure represented. If the Recognins are new protein products whosein situ structural equivalents represent respiratory functions, andthese proteins relate as is now shown to malignancy, then an opening hasbeen found to solve one of the great puzzles of cancer--that is, how arethe energetics satisfied for these voracious, rapidly reproducingmalignant cells?

Aside from the theoretical importance of this discover the data above onthe increase in the percentage of Malignin in more malignant cells, andthe data provided below indicating that Anti-Malignin now only attachesto Malignin-like chemical grouping of the cancer cell, but having soattached is cytotoxic to the cells, come together meaningfully. If themalignin-like in situ compounds in cancer cells are respiratoryproteins, since the Anti-Malignin products of this invention attachpreferentially to these in situ compounds, then if functionalrespiratory groups are involved in this attachment, it is easy tounderstand how this results in death of the cancer cells.

The therapeutic possibilities for the Anti-Malignins, and other similarchemoreciprocals, are greatly strengthened by this structuralinformation on Malignin and Astrocytin, as well as on the demonstratedrelationship of the amount of Malignin to the degree of malignancy.

The molecular weights of the four recognins described with particularityherein, i.e., Astrocytin, Malignin, Recognin M and Recognin L,calculated from their amino acid composition, are close to each other.Thin layer gel chromatography with Sephadex G200 (Pharmacia) gave amolecular weight for both Recognin L and Recognin M of approximately8,000, the same as that obtained with Astrocytin. Since the calculatedmolecular weight of Astrocytin, based upon quantitative amino acidanalysis, is 9,690, the lower apparent molecular weight of 8,000obtained on this layer chrometrography may be, as in the case of MCF-7and P₃ G, a function of the slightly lower net acidic charge of thesethree polypeptides compared to that of Malignin. The molar compositionof the constituent amino acids of the four recognins also are in closeagreement with each other and different from all other knownpolypeptides by computer comparison. The fact that the only fourpolypeptides whose compositions are remotely related are phylogenetiallyancient anaerobic oxidation-reduction enzymes has led to the suggestionthat this may be an important clue to the basis of the anaerobicadvantage of malignant cells recognized for many years but whose basishas not previously been defined. Whether the minor differences betweenthe four cancer recognins represent significant structural differencesis as yet unknown. The composition of twelve of the seventeen aminoacids detected is seen to be identical for each of the three productsfrom those cells which were grown in tissue culture. The principledifferences between the composition of Recognin M and Recognin L whencompared to that of Malignin, are a decrease in aspartic acid, glutamicacid, leucine and tyrosine, and an increase in proline, glycine andalanine.

Despite these chemical differences, the four recognins are, to thepresent, indistinguishable immunologically in each mode tested: (1) eachreacts equally well on Ouchterlony double diffusion with anti-maliginantibody; and (2) separate immobilized (bound to solid support) antigenpreparations of each showed them approximately equal in their ability tobind antibody from human serum regardless of the type of cancer thepatient had that is, immobilized Recognin M was observed to bindantibody from the serum of brain glioma patients as well as didimmobilized Malignin.

EXAMPLE 5A Demonstration of Increased Yield, Degree of Malignancy, andProportion of Malignin, by Provision of Greater Volume and Surface AreaDuring Fermentation

Examples 3 through 5 were repeated using 1000 cc. flasks instead of 250cc. flasks. All quantities of reagents were increased threefold.

The yield of the product MALIGNIN after 7 days growth of inoculum wasincreased almost twofold by increasing the space available for malignantcell growth from 250 cc. to 1000 cc. Table IV shows the yield of totalprotein in mg. and the MALIGNIN produced as a percentage of aid totalprotein for successive generations of fermentation cultures in each sizeflask. Using 250 cc. flasks, the mean total protein produced was 17.5mg. Using 1000 cc. flasks, the mean total protein produced was 40.4 mg.

Surprisingly, as the amount of malignant cell growth (degree ofmalignancy) was increased per seven-day growth period by providinggreater space and surface for cell growth, the amount of MALIGNINproduced, as a percentage of total protein which is MALIGNIN increasedfrom a mean of 10.7 percent using 250 cc. flasks to a mean of 28.3percent using 1000 cc. flasks for a constant growth period of sevendays.

The relationship between the proportion of MALIGNIN produced, and thedegree of malignancy (i.e., as a function of the amount of malignantcell growth in vitro in seven days, measured as total protein produced)is shown in FIG. 4.

                  TABLE IV                                                        ______________________________________                                        Improved Yields in Successive Generations                                     of Fermentation Culture Production of Malignin                                            Malignin,                                                                              Total Protein,                                           Flask Size  mg.      mg.         % Malignin                                   ______________________________________                                         250 cc.    .33      6.4           5.1                                        "           .16      6.7           2.4                                        "           .21      8.9           2.4                                        "           1.3      26.3          4.8                                        "           1.4      21.6          6.4                                        "           2.6      17.9          14.4                                       "           1.8      16.4          10.7                                       "           1.3      13.4          9.8                                        "           2.0      17.8          11.3                                       "           2.3      18.9          12.0                                       "           2.1      19.4          10.8                                       "           1.6      13.8          11.6                                       "           2.2      15.1          14.6                                       "           4.4      21.6          20.4                                       "           3.3      14.0          23.2                                       "           2.2      23.0          9.7                                        "           2.1      23.2          9.0                                        "           2.8      22.3          12.5                                       "           2.4      18.9          12.7                                       "           2.4      24.5          9.8                                                    Mean     17.5    mg.   10.7%                                      1000 cc.    9.8      41.3          23.6                                       "           7.2      25.4          28.4                                       "           5.9      24.9          23.6                                       "           11.7     37.5          31.2                                       "           13.3     44.8          29.8                                       "           16.5     56.5          29.4                                       "           9.5      41.3          22.9                                       "           10.7     38.8          27.5                                       "           12.5     41.6          29.9                                       "           13.3     46.7          29.4                                       "           11.6     45.2          25.7                                                   Mean     40.4    mg.   28.3%                                      ______________________________________                                    

FIG. 4 shows that the increased flask size results in approximatelytrebled proportions of MALIGNIN product.

FIG. 4 also demonstrates a relationship between MALIGNIN and malignancy.The interrupted line represents an ideal linear relationship. Thisdemonstrated relationship is clearly not trivial since the proportion ofMALIGNIN present increases as the cells become more unrestrained(pathological) in their growth. The normal in situ Recognin functionrelates, as previously stated, to the contact inhibition of growth ofcells. The more pathological the growth of the malignant cells, the lesscontact inhibition operates, and the more MALIGNIN becomes thepredominant protein.

Example 5A demonstrates that growth of artificial cancer cell culture inlarge size growth containers unexpectedly results in increasedproportion of MALIGNIN produced, that is, in an increase in thepercentage of total protein produced which is MALIGNIN. As used in thisapplication, a large size growth container means one in which the ratioof the container volume to the volume of total medium which cellsutilized in accordance with the methods of Example 3 is greater thanabout 8:1, for example, 7:1 to 10:1. Example 5A illustrates a ratio ofabout 8:1.

EXAMPLE 6 Production of TARGET Reagents from RECOGNINS

ASTROCYTIN, prepared as in EXAMPLE 2 above, or MALIGNIN, prepared as anEXAMPLE 5 above, or Recognin M or Recognin L is complexed with a carrierto produce TARGET reagent.

In the preferred embodiment, which is applicable to any RECOGNIN,ASTROCYTIN, MALIGNIN, RECOGNIN L or RECOGNIN M, is dissolved in 0.15MNaH₂ PO₄ -citrate buffer, pH 4.0. A bromoacetyl-resin, for example,bromoacetylcellulose (BAC) having 1.0 to 1.5 milliequivalents Br pergram of cellulose, stored in the cold, is prepared in 0.15M NaH₂ PO₄buffer, pH 7.2. Convert the buffer to pH 4 by pouring off the pH 7.2buffer solution and adding 0.15M NaH₂ PO₃ -citrate buffer, pH 4.0. TheRECOGNIN solution and the BAC solution are stirred together (10:1 BAC toRECOGNIN ratio) for 30 hours at room temperature, then centrifuged.

It is preferred that all sites on the BAC which are available to bind toRECOGNIN be bound. This may be accomplished as follows. The supernatantfrom the immediately preceding step is lyophilized and the proteincontent determined to indicate the amount of RECOGNIN not yet complexedto BAC. The complexed BAC-RECOGNIN is resuspended in 0.1M bicarbonatebuffer pH 8.9, stirred 24 hours at 4° to permit the formation ofchemical bonds between the BAC and the RECOGNIN. After the 24 hours, thesuspension is centrifuged and supernatant analyzed for protein. Thecomplexed BACRECOGNIN is now resuspended in 0.05M aminoethanol-0.1Mbicarbonate buffer pH 8.9 in order to block any unreacted bromine. Thesuspension is centrifuged, and the supernatant is kept but not analyzedbecause of the presence of aminoethanol. Removal of all unbound RECOGNINis then accomplished by centrifugation and resuspension for threewashings in 0.15M NaCl until no absorbance is measured on thespectrophotometer at 266 mu. The BAC-RECOGNIN complex is now stirred in8M urea for 2 hours at 38° C., centrifuged, then washed (three timesusually suffices) with 8M urea until no absorbance is shown in thewashings at 266 mu. The complex is then stirred at 37° C. in 0.25Macetic acid for 2 hours to demonstrate its stability. Centrifuge and theread supernatant at 266 mu--no absorbance should be present. Thischemically complexed BAC-RECOGNIN is therefore stable and can now beused as a reagent in the methods described below; in this stable reagentform it is referred to as a synthetically produced complex whosephysical and chemical properties mimic the stable cellbound precursor ofthe RECOGNIN when it is in a potential reactive state with serumcomponents. For storing, TARGET reagent is centrifuged and washed untilneutralized with 0.15M NaH₂ PO₄ buffer pH 7.2.

TARGET reagents may be prepared from bromoacetyl liganded carriers otherthan cellulose, such as bromoacetylated resins or even filter paper.

EXAMPLE 7 Production of antisera to Astrocytin, Malignin, and TARGET

Antisera to Astrocytin, Malignin, or TARGET reagents may be produced byinducing an antibody response in a mammal to them. The followingprocedure has been found to be satisfactory.

One mg. of RECOGNIN is injected into the toe pads of white male rabbitswith standard Freund's adjuvant, and then the same injection is madeintraperitoneally one week later, again intraperitoneally ten days and,if necessary, three weeks later. Specific antibodies may be detected inthe blood serum of these rabbits as early as one week to ten days afterthe first injection. The same procedure is followed for TARGET antigenby injecting that amount of TARGET which contains 1 mg. of Astrocytin orMalignin as determined by Folin-Lowry determination of protein.

The specific antibody to Astrocytin is named AntiAstrocytin. Thespecific antibody to Malignin is named Anti-Malignin. Similarly, thespecific antibody to TARGET reagent is named Anti-Target.

These antibodies show clearly on standard Ouchterlony gel diffusiontests for antigen-antibody reactions with specific single sharp reactionlines produced with their specific antigen.

The presence of specific antibodies in serum can also be tested by thestandard quantitative precipitin test for antigen-antibody reactions.Good quantitative precipitin curves are obtained and the micrograms ofspecific antibody can be calculated therefrom.

Further evidence of the presence of specific antibodies in serum can beobtained by absorption of the specific antibody Anti-Astrocytin ontoBromoacetyl-cellulose-Astrocytin (BAC-Astrocytin) prepared above. Theantiserum containing specific Anti-Astrocytin can be reacted withBAC-Astrocytin. When the serum is passed over BAC-Astrocytin only thespecific antibodies to Astrocytin bind to their specific antigenAstrocytin. Since Astrocytin is covalently bound toBromoacetyl-cellulose the specific antibody, Anti-Astrocytin, is nowbound to BAC-Astrocytin to produce BAC-Astrocytin-Anti-Astrocytin(BACA-Anti-Astrocytin). This is proved by testing the remainder of theserum which is washed free from BAC-Astrocytin. On standard Ouchterlonydiffusion no antibodies now remain in the serum which will react withAstrocytin. It is therefore concluded that all specific antibodies(Anti-Astrocytin) previously shown to be present in the serum, have beenabsorbed to BAC-Astrocytin. Furthermore, when Anti-Astrocytin isreleased from its binding to BAC-Astrocytin it is thereby isolated freeof all contaminating antibodies. This release of Anti-Astrocytin may beaccomplished by washing the BACA-Anti-Astrocytin coupled with 0.25Maccetic acid (4° C., 2 hrs.) which has been shown above not to break theBAC-Astrocytin bond.

The antibodies to TARGET show clearly on standard Ouchterlony geldiffusion tests for antigen-antibody reactions with specific singlereaction lines produced with TARGET which show a line of identity withthe line of reaction to ANTI-ASTROCYTIN or ANTI-MALIGNIN antisera (i.e.,that produced to the injection of ASTROCYTIN or MALIGNIN themselves).Some rabbits, it has been noted, have levels of ANTI-TARGET in theirblood prior to being injected with TARGET. These ANTI-TARGET substances,when reacted specifically with TARGET reagent as to be described intests of human sera, lead to the production of approximately equivalentamounts of the two types of TAG, S-TAG and F-TAG (see later EXAMPLES).

EXAMPLE 8 Detection of Malignant Tumors by Quantitative Production invitro of TARGET-ATTACHING-GLOBULINS (TAG) from Biological Fluids

TARGET reagent prepared in accordance with EXAMPLE 6 is washed to removeany unbound RECOGNIN which may be present due to deterioration. Thefollowing procedure is satisfactory. TARGET reagent is stirred for twohours at 37° C. with acetic acid, centrifuged, the supernatant decanted,and the optical density of the supernatant read at 266mu. If there isany absorbance, this wash is repeated until no further material issolubilized. The TARGET is then resuspended is phosphate bufferedsaline, pH 7.2. (Standard S-TAG and F-TAG purified from previousreactions of human serum by the procedure described below can be used ifavailable, as reference standards to test the TARGET reagent, as canwhole rabbit serum which has been determined to contain S-TAG and F-TAGby other TARGET preparations).

The Slow-Binding (S-TAG) determination is performed as follows: Frozenserum stored more than a few days should not be used. Serum is carefullyprepared from freshly obtained whole blood or other body fluid bystandard procedures in the art. The following procedure has been foundto be satisfactory. Blood is allowed to clot by standing for 2 hours atroom temperature in a glass test tube. The clots are separated from thewalls with a glass stirring rod, and the blood allowed to stand at 4° C.for a minimum of 2 hours (or overnight). The clots are separated theserum by centrifuging at 20,000 rpm at 4° C. for 45 minutes. The serumis decanted into a centrifuge tube and centrifuged again at 2000 rpm at4° C. for 45 minutes. The serum is decanted and a 1% Solution ofMethiolate (1 g. in 95 ml. water and 5 ml. 0.2M bicarbonate buffer pH10) is added to the extent of 1% of the volume and serum.

Serum samples, prepared by the above or other procedures, of 0,2 ml.each are added to each of 0.25 ml. aliquots of TARGET suspension reagentcontaining 100-200 micrograms of RECOGNIN per 0.25 ml. TARGET reagent,in duplicate determination. The suspension is mixed at 4° C. in a mannerto avoid pellet formation. For example, a small rubber cap rapidlyshaken may be used for 1-2 seconds and then, with the tubes slightlyslanted, they may be shaken in a Thomas shaker for about 2 hours ormore. The TARGET reagent and protein bound to it are separated from theserum. One of the procedures which has been found to be satisfactory isthe following. The tubes are then centrifuged at 2000 rpm for 20 minutesat 4° C., the supernatant decanted, the pellet which is formed bycentrifugation washed 3 times by remixing and shaking at roomtemperature with 0.2-0.3 ml. of 0.15M. Saline, centrifuged and thesupernatants discarded.

The protein which remains attached to the TARGET is cleaved therefromand quantitatively determined. For example, 0.2 ml. of 0.25M acetic acidis needed, the suspension shaken for 1 to 2 hours in a 37° C. incubator.The tubes are centrifuged at 2000 rpm at 4° C. for 30 minutes. Thesupernatant is carefully decanted to avoid transferring particles andthe optical density of the supernatant is read at 280 mu. The value ofthe optical density is divided by a factor of 1.46 for results inmicrograms per ml. serum protein (S-TAG). Duplicate determinationsshould not vary more than 5%. Any other procedure effective fordetermining protein content may be used, such as Folin-Lowrydetermination, but standards must be specified to determine the range ofcontrol and tumor values of S-TAG minus F-TAG concentration.

The Fast-Binding (F-TAG) determination is performed as follows: Frozenserum stored more than a few days should not be used. Serum is carefullyprepared from freshly obtained whole blood or other body fluid bystandard procedures in the art. The procedure given above in thisEXAMPLE for serum preparation is satisfactory.

Serum samples, prepared by the above or other procedures are allowed tostand at 4° C. for 10 minutes less than the total time the S-TAG serumdeterminations were allowed to be in contact with TARGET reagent above[e.g. 1 hour 50 minutes if a "two hour" S-TAG determination was made].This procedure equilibrates the temperature histories of S-TAG and F-TAGdeterminations.

Add 0.2 ml. samples of the temperature equilibrated serum to each of0.25 ml. aliquots of TARGET suspension reagent containing 100-200micrograms of RECOGNIN per 0.25 ml. TARGET reagent, in duplicatedetermination. The suspension is then mixed at 40° C. for approximately10 minutes in a manner to avoid pellet formation. For example, a smallrubber cap rapidly shaken may be used for 1-2 seconds and then, with thetubes slightly slanted, they may be shaked in a Thomas shaker forapproximately 10 minutes. The TARGET reagent and protein bound to it areseparated from the serum. One of the procedures which has been found tobe satisfactory is the following. The tubes are then centrifuged at 2000rpm for 20 minutes at 4° C., the supernatant decanted, the pellet whichis formed by centrifugation washed 3 times by remixing and shaking atroom temperature with 0.2-0.3 ml. of 0.15M Saline, centrifuged and thesupernatants discarded.

The protein which remains attached to the TARGET is cleaved therefromand quantitatively determined. The procedure described above in thisEXAMPLE for determining S-TAG concentration is satisfactory. Any otherprocedure effective for determining protein content may be used, such asFolin-Lowry determination, but standards must be specified to determinethe range of control and tumor values of S-TAG minus F-TAGconcentration.

The final results are expressed as TAG micrograms per ml. of Serum, andequal S-TAG minus F-TAG. TAG values in nonbrain-tumor patients and othercontrols currently range from zero (or a negative number) to 135 or 140micrograms per ml. of serum. In any result over 100 ug/ml., a repeatdetermination is indicated. Some other tumors may yield high TAG values,especially if secondary (metastic) tumors are present in the brain. TAGvalues in brain tumor patients currently range from 136 to 500 or moremicrograms per ml. of serum. In the first "blind" study of 50 bloodsamples conducted according to the procedures of this EXAMPLE utilizingTARGET reagent prepared from Astrocytin and bromoacetylcellulose, 11 of11 brain tumors and 28 of 32 normals were correctly identified. One ofthe 4 supposed normals (i.e., non-brain tumor controls) turned out tohave a cancer of the thyroid gland which had apparently beensuccessfully treated some years before. The three remaining normals wereindividuals aged 60-70 who were in poor health, possibly havingnondiagnosed cancer. Of the remaining 7 samples, three out of threecases of Hodgkin's Disease were correctly identified; one sample in thetumor range (136-500 ug. TAG/ml.) corresponded to a patient having asevere gliosis, and three samples in the non-tumor range (0-135 ug.TAG/ml.) corresponded to patients having respectively, and intercranialmass diagnosis uncertain but non-tumor, and osteosarcoma (non-braintumor) and a melanotic sarcoma (non-brain tumor).

A subsequent study conducted according to the procedure of this exampleutilizing TARGET reagent prepared from Malignan and bromoacetylcellulosecorrectly differentiated three out of three malignant brain tumors andall normals.

A still further study conducted according to the procedure of thisexample extended the total number of human serum specimens tested from50 to 114. Half of the serum specimens were tested with Malignin-derivedTARGET reagent and half were tested with the correspondingAstrocytin-derived reagent. The results of this study is summarized inTable V.

EXAMPLE 9 Diagnosis of Tumor Cells by Immunofluorenscence

The compounds Anti-Astrocytin, Anti-Malignin, and S-TAG have been shownto attach preferentially to tumor cells. This specificity permits use ofthree compounds to diagnose tumor cells in histology sections byconjugating dyes or radioactive substances to Anti-Astrocytin,Anti-Malignin, or S-TAG. Standard labeling techniques may then be used.A procedure using S-TAG is as follows.

One procedure which has been found satisfactory is a modified St. Marieprocedure. Human brain tumor specimens are frozen and 5 micron thicksections cut. They are stored in a moist container at minus 70° C. for 4to 8 weeks before staining. The conjugate may be a standard antiserumsuch as goat anti-rabbit conjugate. The conjugate is labeled bytechniques known in the art with fluorescent or other labelingsubstance. Fluorescein labeled goat anti-rabbit conjugate ascommercially-available may be used. The fluorescent technique used was astandard one in which a 1:200 to 1:400 solution of TAG is incubated forabout 30 minutes or more on the tumor section, followed by washes toremove unattached TAG. Three washes with phosphate buffered saline hasbeen found satisfactory. Conjugate incubation with fluorescein-labeledconjugate followed by washes is then performed, followed by microscopicinspection. Normal cells and their processes fail to stain both in tumorsections and in control sections of normal non-tumor brain. Fluorescenceis brightly present in tumor glial cells and their processes.

                                      TABLE V                                     __________________________________________________________________________                            Malignant                                                                             Malignant                                                     Malignant                                                                             Other Tumors,                                                                         Other Tumors,                                                                         Uncertain                                             Brain Tumors,                                                                         Brain   No Brain                                                                              Cerebral                              Normals*        Primary Secondaries                                                                           Secondaries                                                                           Diagnosis                             Serum                                                                             Serum                                                                             Serum                                                                             Serum                                                                             Serum   Serum   Serum   Serum                                 TAG TAG TAG TAG TAG     TAG     TAG     TAG                                   ug/ml                                                                             ug/ml                                                                             ug/ml                                                                             ug/ml                                                                             ug/ml   ug/ml   ug/ml   ug/ml                                 __________________________________________________________________________    124 19  54  65  459     270      36.sup.4                                                                             165%                                  113 55  27  113 397     257      31.sup.5                                                                             144.sup.9                             105 51  41  130 236     188     442.sup.14                                                                             13.sup.9                             130 82  21  79  137     205     288.sup.14                                                                            .sup. 209.sup.10                      127 44  27  61  298     .sup. 157.sup.7 .sup.  75.sup.10                       38 127 21  123 397                     .sup. 184.sup.11                      100 31   0  14  241                     .sup.  27.sup.11                      125  0  14  20  241                     .sup. 110.sup.12                       30 125 62  41  217                     .sup. 192.sup.15                      .sup. 250.sup.1                                                                   118 38  34  147                                                            39 89  93  93  127                                                           .sup. 363.sup.1                                                                   .sup. 99.sup.6                                                                    21  48  185                                                            4  .sup. 13.sup.2                                                                     0  20  253                                                            31 270.sup.3                                                                         120 82  253                                                            42  7  16  20  565                                                            34 58  20  55  277                                                            76 24  113  0  137                                                            48 62  72      .sup.  78.sup.13                                               85 89          138                                                               89          650                                                                           160                                                           __________________________________________________________________________     *Includes normals, nontumor medical and surgical disorders. 1very ill;        undiagnosed, 2Extra brain intracranial mass, undiagnosed, 3Marked gliosis     4Malignant melanoma, 5Osteosar-coma, 6Brain cyst fluid, 7Adenocarcinoma o     colon, 8Gastrectomy, 9Headaches, 10Emphysema, 11Polymyalgia, 12Colon          cancer, 13Convulsions, 14Cancer of prostrate, secondaries to bone,            15Clinically "normal", 18Months earlier, when this abnormal serum TAG         obtained: Now developed sever headaches, loss of smell and taste.        

EXAMPLE 10 Demonstration that Anti-Astrocytin, Anti-Malignin and S-TAGare Cytotoxic to Tumor Cells Growing in Tissue Culture

Standard tests for determining cytoxicity may be used. Generally, thenumber of cells in a fixed counting chamber, usually arranged to containabout 100 live cells, is counted. These cells are then treated with theagent being tested and the number of cells which are still alive iscounted.

In a standard test of cytotoxicity of S-TAG Solution obtained inaccordance with the methods of EXAMPLE 8 against cells in tissue culturederived from a patient with a glioblastoma Grade III-IV, wellcharacterized as of glial origin, S-TAG produced death of all cells inthe counting chamber even when in high dilution of 1:100 and 1:1000,representing as little as 0.2 and 0.02 ug. of S-TAG per ml. of solution.Similar results are obtained with high dilutions of AntiAstricytin andAnti-Malignin.

Both the specificity exhibited in EXAMPLE 9 and the cytotoxicitydemonstrated in EXAMPLE 10 are highly relevant to the therapeuticpossibilities of Anti-Astrocytin, Anti-Malignin and S-TAG for braintumors in man. These therapeutic users are in addition to the practicaldiagnostic potential of both of these phenomena for tumor tissue removedat operation but requiring diagnosis by histology already demonstratedherein.

EXAMPLE 11 Hydrolytic Cleavage of RECOGNINS

A solution of RECOGNIN, in this case either Astrocytin or Malignin at pHbetween 1 and 2 is allowed to stand in the cold. After 7 to 14 days, TLGchromatography shows the product to have been reduced in molecularweight by approximately 200. When the solution is allowed to standlonger, further units of approximately 200 molecular weight are cleavedevery 7 to 10 days. Thus with Astrocytin the molecular weight is reducedfrom 8,000, and with MALIGNIN the molecular weight is reduced from10,000, in each case by units of approximately 200 sequentially.

The physiochemical specificities of Astocytin are retained by eachproduct down to approximately 4,000 molecular weight. The physiochemicalspecificities of Malignin are retained by each product down toapproximately 5,000 molecuar weight. This is shown by Ouchterlony geldiffusion tests against Anti-Astrocytin and Anti-Malignin, respectively.

This cleavage can also be accomplished exzymatically, as with tryspinand other proteindases, with similar results.

The molecular weights of these compounds prepared by hydrolytic cleavageof RECOGNINS may be approximately defined by the following formulae:

For products having the physiochemical specificities of Astrocytin;400+200 x=Y.

For products having the physiochemical specificities of Malignin;5000+200 x=Y wherein Y is the molecular weight of the product and X isan integer from 0 to 19.

EXAMPLE 12 Production of Artificial Tissue or Organ with RECOGNINS

A rigid walled tube of plastic, metal, or other suitable rigid materialis dipped in or impregnated with a highly concentrated, [i.e., 10mg./ml.] viscous solution of RECOGNIN, in this case either Astrocytin orMalignin, until all surfaces are fully coated with the RECOGNIN.Alternately, RECOGNIN solution is passed through and around the tubeunder pressure until all surfaces are fully coated. The tube is thendried in air or in vacuo, or lyophilized. The process of coating isrepeated several times in order to build up multiple molecular layers ofRECOGNIN coating.

The tube is now ready to be placed in a cavity or in a tissue whichcontains Astrocytin or Malignin-like precursors in the neighboringtissue or fluid of a living mammal. This artificial tissue or organ maybe used to minimize or eliminate reaction which foreign substanceswithout RECOGNIN coating would incite.

Artificial tissues or organs of other geometries may similarly beproduced.

EXAMPLE 13

Malignin is useful in a serum diagnostic test for the detection of awide variety of tumors, including non-brain malignancies. The results ofdeterminations on individual sera, and remarks related thereto, arereported in the following table.

                                      TABLE 1                                     __________________________________________________________________________    ANTI-MALIGNAN ANTIBODY DETERMINATION IN NON-BRAIN CANCERS                     WITH MALIGNAN-BASED TARGET REAGENT                                                     Number                                                                            Number    Number          Antibody bound, as Protein Com-                 of  Abnor-                                                                             Number                                                                             in        %     plexes in micrograms/ml Serum          Non-Brain.sup.(a)                                                                      Serums                                                                            mally                                                                              Border-                                                                            Normal                                                                             %    Elevated +                                                                          S-TAG    Net TAG                       Malignancies                                                                           Studied                                                                           Elevated                                                                           line Range.sup.(c)                                                                      Elevated                                                                           Borderline                                                                          Mean                                                                              (±S.D.)                                                                         Mean                                                                              (±S.D.)                __________________________________________________________________________    Carcinoma of:                                                                 Lung     6   5         1    83.3 83.3                                         Breast.sup.(g)                                                                         10  10             100.0                                                                              100.0                                        Colon.sup.(g)                                                                          7   7              100.0                                                                              100.0                                        Rectum.sup.(g)                                                                         6   5         1    83.3 83.3                                         Prostate 2   2              100.0                                                                              100.0                                        Bladder  1   1              100.0                                                                              100.0                                        Ovary    4   2    1    1    50.0 75.0                                         Kidney   1   1              100.0                                                                              100.0                                        Thyroid  1   1              100.0                                                                              100.0                                        Undiffer-                                                                              2   2              100.0                                                                              100.0                                        entiated                                                                      Lymphoma 10  9         1    90.0 90.0                                         Hodgkins'                                                                              6   5    1         83.3 100.0                                        Disease                                                                       Multiple 6   6              100.0                                                                              100.0                                        Myeloma                                                                       Acute                                                                         Myelogenous                                                                            2   2              100.0                                                                              100.0                                        Leukemia                                                                      Chronic                                                                       Myelogenous                                                                            5   5              100.0                                                                              100.0                                        Leukemia.sup.(c)                                                              Chronic                                                                       Lymphocytic                                                                            3   2    1         66.6 100.0                                        Leukemia.sup.(c)                                                              Osteogenic                                                                             1             1    0    0                                            Sarcoma                                                                       Melanotic                                                                              4   3         1    75.0 75.0                                         Sarcoma                                                                       Total Non-Brain                                                                        77  68   3    6    88.3 92.2  482.5                                                                             (±202.8)                                                                        168.1                                                                             (±145.9)               Malignancies                                                                  Brain                                                                         Cancer.sup.(d,g)                                                                       85  80   1    4    94.1 95.3  456.3                                                                             (±146.9)                                                                        183.4                                                                             (±116.3)               Total                                                                         Malignancies                                                                           162 148  4    10   91.4 93.8                                         Non-Malignant                                                                          51  3    --.sup.(e)                                                                         48   5.9  --    259.8                                                                             (±115.8)                                                                        59.5                                                                              (±66.0)                Medical and                                                                   Surgical                                                                      Disorders                                                                     Normal   77  3    --.sup.(f)                                                                         74   3.9  --    280.3                                                                             (±79.5)                                                                         60.2                                                                              (±46.9)                Total Number 290; % correct = 91.4; 94.5% including borderline                __________________________________________________________________________    values.                                                                        Legend:                                                                       S-TAG is the micrograms per ml. serum of antibody bound in 2 hours            incubation; FTAG is that bound in 10 minutes incubation; Net TAG =            (STAG)-(F-TAG). Net TAG normal values 0 to 134 micrograms per ml. serum.      Elevated values: Net TAG 135 micrograms per ml. serum or greater, or STAG     greater than 400 micrograms per ml. even if high FTAG value results in Ne     TAG less than 135 micrograms per ml. Borderline values 100 to 134             micrograms per ml. The values for STAG were significantly different for       Gps. I and II as compared with Gps. III and IV at a level of p less than      0.000001 and for Net TAG at a level of p less than 0.000001. All of these     determinations were performed blind. Only those cases in which the            clinical source of the serum was known, the clinicalpathological diagnosi     established, and the serum received in good condition frozen in dry ice       not longer than 48 hours from the time it was drawn are included in the       study; 270 specimens had to be excluded on these bases. .sup.(a) Seven of     these determinations performed with Astrocytinbased TARGET reagent: 1         cancer of colon, 1 cancer of thyroid, 3 Hodgkins' Disease, 1 osteogenic       sarcoma, 1 melanotic sarcoma. .sup.(b) polycythemia vera. .sup.(c)            Terminal cases: 1 leukemia excluded, some additional may also by excluded     .sup.(d) Eleven of these determinations performed with Astrocytinbased        TARGET reagent. .sup.(e) Six cases originally borderline, then normal whe     repeated; one sickle cell anemia in crisis with renal insufficiency, 1        case of obesity, 1 of gout. .sup.(f) Eleven cases originally borderline,      then normal when repeated. .sup.(g) Two cases of breast cancer, 1 of          rectum and 1 of brain demonstrated normal values of serum TAG and 2 colon     cancer, borderline values, on serial determination during course of cance                                                                              

EXAMPLE 14 Detection of Non-Brain Malignant Cells With FluorescentSignal From TAG

The uses of TAG products coupled with a signal emitter such as a dye ora radioactive label to detect cancer cells is described previously andherein. In this EXAMPLE 14, the detection of non-brain malignant cellsis described.

As described in EXAMPLE 8 utilizing human serum; in the determination ofTAG, after the anti-malignin antibody was bound to the immobilizedantigen and non-bound serum proteins washed away, the antibody wascloven from the binding with 0.25M acetic acid at 37° C. for 2 hours andthe TARGET reagent separated from it by centrifugation. The TAG antibodysolution was quantitated by means of its absorption at 280 μ. The TAGsolutions were stored at -20° C., then thawed and combined, brought topH 7 by titration with 6N NaOH, dialyzed against phosphate bufferedsaline pH 7, filtered and concentrated on Millipore Pellicon 1000membranes, centrifuged to clear insoluble protein and the immuneglobulin complexes concentrated and freed of immunologically non-activecompounds by Cellex D and Blue Sepharose CL6B (Pharmacia)chromatography. This human anti-malignin antibody reacts with antihumangamma globulin in Ouchterlony double diffusion. When TAG is used withfluorescein conjugated to anti-human gamma globulin in standard doublelayer Coons immunofluorescence it stains malignant glia, breastcarcinoma, ovarian carcinoma, adenocarcinoma of colon, and other typesof cancer cells in postoperative and biopsy tissue sections, as well asin human sputum, bronchial washings, pleural effusion fluid, gastricaspirate and bladder urine. The concentration of protein in TAG whichyield clear fluorescence when controls are negative, is 1 to 10 μg persection.

The production of a "purified" TAG was undertaken by reacting the serafrom patients with a variety of cancers withbromoacetylcellulose-MALIGNIN by methods earlier described (EXAMPLE 8).The antibody bound in this reaction was cleaved with 0.25M acetic acid,quantified by measurement at O.D. 280 using a conversion factor of 1.46for gamma globulin, frozen and stored at -20° C. This antibody was foundto contain immunoglobulin as determined by anti-human gamma-globulinantiserum specific for gamma chains (BioRad Laboratories, Inc.) and withanti-FAB and anti-Fc fragments (Miles Laboratories). It also reacts withrabbit anti-human albumin (BioRad Laboratories).

It was found that whereas 10 to 50 micrograms of protein TAG arerequired to produce specific immunofluorescent staining of cells whichcontain Malignin, only 1 to 10 micrograms of purified protein TAG arerequired for this specific staining in all sections, and in a few, lessthan one microgram has been found to suffice.

It was found that the most active preparation of purified TAG is thatwhich is eluted with the highest ionic strength elution, i.e., from0.15M to 1.5M. Any method of production which uses this fact is useful;three preferred methods are given below.

Method I--Fractionation of TAG by chromatography with DEAE cellulose(Cellex D, BioRad Laboratories) was first employed with step-wiseelution with increasing ionic strength and decreasing pH, the samesequence of eluants as that given in Example I for the production ofCrude Astrocytin-Precursor-Containing Fraction. Good separation wasobtained of the bulk of the protein into three fractions, Peak Iobtained with Solution 1 (see Example 1) and Peak II obtained withSolution 6 and Solution 7. Ouchterlony double diffusion showed the TAGin Peak I still to contain appreciable protein with albumin mobility,and while Peak II contained most of the albumin, appreciable IgG couldbe detected. Rechromatography of Peak I gave a progressively pure IgGuntil, after the seventh chromatography, essentially no albumin (lessthan 3%) could be detected by Ouchterlony gel diffusion in which 5 to 10micrograms of human albumin was detectable with rabbit anti-humanalbumin. The IgG so obtained was prone to denaturation and loss ofimmunological reactivity after a few days standing at 0°-5° C.

Method II--A second fractionation of TAG was made with chromatography onSepharose CL-6B (Pharmacia, Inc.) starting with low molarity buffer(0.0005M phosphate) and proceeding in two steps of 0.15M and 1.5M toelute the balance of the protein. As with the Cellex D, one passage wasfound to be inadequate to separate, and recycling slowly improved theproduct. Once again, the most active fraction vis-a-vis anti-maligninantibody was in the 1.5M fraction.

Method III--Chromatography with Sepharose CL-6B next to the glassfritted disc and Cellex D layered above the Sepharose proved to be themost satisfactory method.

The graphical representation in FIG. 1 shows the fractions obtained onchromatography of TAG utilizing Method III. After the first eluate of200 mls., 50 ml. or smaller sub-fractions were collected. The proteincontent of each eluate was determined by the optical density at 200 muwith a uniform factor of 1.46 based on gamma globulin used to convert tomicrograms for calculating recoveries. The absolute amount of proteinrequires correction in those fractions in which there is appreciablealbumin. The points at which the stepwise solvent changes were made areindicated by arrows. The subfractions are designated by Roman Numerals Ithrough VIII.

The solvents corresponding to letters A-F at the arrows were as follows:

A--0.01M TRIS (pH 7.2)

B--0.05M TRIS with 0.1M NaCl (pH 7.2)

C--PBS, 0.11M NaCl (pH 7.2)

D--PBS, 0.165M NaCl (pH 7.2)

E--PBS, 0.33M NaCl (pH 7.2)

F--0.05M TRIS, 1.5M NaCl (pH 7.2)

In the following Table are shown the recoveries from each fraction, asemi-quantitative determination in each of the gamma-globulin andalbumin in each, as well as the activity of each fraction in theimmunofluorescent staining of cancer cells. (The plus sign indicatesreaction, zerp mp reaction and plus/minus reaction in some cases).

                                      TABLE                                       __________________________________________________________________________    Fraction   I   II III                                                                              IV V   VI  VII VIII                                      __________________________________________________________________________    Recovered                                                                     ug         2,877                                                                             1,140                                                                            2,351                                                                            2,942                                                                            1,808                                                                             2,230                                                                             2,125                                                                             7,477                                     %          12.5                                                                              5.0                                                                              10.2                                                                             12.8                                                                             7.9 9.7 9.3 32.6                                      Immunodiffusion                                                               Against:                                                                      Anti-human +++ ++ ++ +  o   +   ++  +++                                       IgG. specific                                                                 for gamma chains                                                              Anti-human +   +  +  ++ +++ +++ +++ +++                                       aluminum                                                                      Anti-Fab   +   ++ o  ++ +   o   ++  ++                                        Anti-Fc    ++  ++ +  ++ +   o   +   o                                         Immunofluorescence                                                                       ±                                                                              ±                                                                             ±                                                                             ±                                                                             ±                                                                              ±                                                                              ±                                                                              +++                                       __________________________________________________________________________

FIG. 2 is a photograph showing the line of reaction between anti-humangamma globulin specific for gamma chains for each of Fractions I and IIfrom above (left and below in photograph).

FIGS. 3a and 3b are photographs showing the use of TAG (Fraction VIIIfrom above) to stain non-brain malignant cells. FIG. 3a is a stain ofbronchogenic carcinoma cells in the bronchial washings of a patient;FIG. 3b is a stain of lymphoma cells in the pleural fluid of a patient.Non-cancer cells do not fluoresce. The TAG (1 to 10 ug in 0.1 mlphosphate buffered saline (PBS)) is applied to the surface of packedcells on a glass slide incubated 30 minutes, washed three times with PBSand then layered with fluoroscein-conjugated anti-human IgG diluteduntil non-malignant control tissues give essentially no fluorescence.The cells are visualized with a Zeiss fluorescent microscope using atungsten lamp and filters BG 23, BG 12 and 500.

EXAMPLE 9B Detection of Cancer Cells With Radioisotope Signal From Tag

In this Example, the feasibility of attaching a radioactive label to TAGis demonstrated. Second, the injection into animals of thisradio-labeled TAG has been accomplished and shown to be safe andeffective. Third, the radio-labeled TAG localized preferentially in thecancer tissue when compared to normal tissue, thus indicating that thespecificity previously demonstrated in vitro of the preference forcancer cells which is conveyed by the use of specific anti-Malignin TAGproducts is confirmed in vivo.

The Labeling of TAG with 99m Technetium (^(99m) Tc)

Procedure for Labeling

1. Two preparations of TAG were used, here designated TAG-1 and TAG-2.TAG-1 and TAG-2 (concentration of each 0.4 mg/0.5 ml) were added toseparate sterile evacuated vials.

2. To each vial was added 0.1 ml of a stannous chloride solution (10 mgSnCl₂. 2 H₂ O in 100 ml of 0.01N HCl). The vials were mixed for 3-4minutes.

3. 0.1 ml. (6mCi) of ^(99m) Tc-pertechne2tate (sodium salt) was addedand mixed 2-3 minutes.

Procedure for determining labeling efficiency

Samples of the ^(99m) Tc-TAG-1 and ^(99m) Tc-TAG-2 were tested forlabeling efficiency by descending paper chromatography using Watman No.1 paper with 85% methanol as the solvent. A similar study was done withSodium Pertechnetate-^(99m) Tc which acted as a control. After 2 hours,the papers were removed from the chromatography tank and divided in twosections: (1) 1 cm about the origin; (2) the remaining paper up to thesolvent front. Each section was then counted in a gamma wellscintillation counter and its content of radioactivity determined (cpm).

Approximately 50 lambda were plated on each paper strip.

Procedure for Antigen-Antibody Reaction

A portion of the labeled solution was also plated on an Ouchterlony gelplate to determine its ability to react with malignin in theantigen-antibody reaction. After a 3 hour period, the resulting sharpreactive lines were removed from the gel and their content ofradioactivity measured. An equal portion of the gel not involved in thereaction was also removed and its content of radio activity was alsomeasured as background.

Results

Labeling Efficiency

                                      TABLE 1                                     __________________________________________________________________________    Labeling Efficiency of .sup.99m Tc--TAG--1 and .sup.99m Tc--TAG-- 2           COMPOUND SITE ON PAPER                                                                           CPM   %    CHEMICAL SPECIES                                __________________________________________________________________________    NaTcO.sub.4 --99 mTc                                                                   origin    4.94 × 10.sup.5                                                               7.33%                                                                              reduced TcO.sub.4 --                            NaTcO.sub.4 --99 mTc                                                                   solvent front                                                                           6.25 × 10.sup.6                                                               92.67%                                                                             TcO.sup.4 --                                    TAG--1   origin    4.35 × 10.sup.6                                                               98.47%                                                                             TAG--99 mTc                                     TAG--1   solvent front                                                                           6.76 × 10.sup.4                                                               1.53%                                                                              TcO.sub.4 --                                    TAG--2   origin    1.96 × 10.sup.6                                                               98.01%                                                                             TAG--99 mTc                                     TAG--2   solvent front                                                                           3.98 × 10.sup.4                                                               1.99%                                                                              TcO.sub.4 --                                    __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        ANTIGEN-ANTIBODY RESECTION                                                    GEL AREA     COUNTS PER MIN %                                                 ______________________________________                                        TAG--2 line  1.99 × 10.sup.6                                                                        92.04%                                            Background gel                                                                             1.72 × 10.sup.5                                                                        7.96%                                             ______________________________________                                    

Conclusions

The following conclusions were reached relative to the quality controltests employed:

1. ^(99m) Tc-pertechnetate was reduced by stannous chloride to a morereactive oxidation state (+4+5).

2. The reduced pertechnetate labeled both the TAG-1 and TAG-2preparations.

3. The ^(99m) Tc-TAG-2 was tested for its ability to retain its activityand was found to retain its ability to react immunologically.

The Use of Radio Labeled TAG in vivo to Detect Cancer Cells

Wistar rats were injected intracerebrally with Cisgliome tumor cellswhich had had previous passages in rats and in tissue culture. The ratswere observed for the first signs of growing tumor, such as weakness,tremor or unsteadiness. These symptoms first appear seven to 10 daysfrom injection, and with fast growing tumors result in death withinthree to four days in many animals, and one week in all. As soon assymptoms appeared, the animals were injected with labeled TAGintravenously in the tail vein, then the animal anesthetized at varyingtimes, the brain removed, the tumor dissected free of normal brain, andthe radioactivity in each dissected specimen compared.

    ______________________________________                                        Preliminary .sup.99m Tc--TAG experiment                                                      Counts/gm/min.                                                        Sacrifice     Tumor            Normal                                  Animal (hr. post injection)                                                                        wt., mg.  Tumor  Brain                                   ______________________________________                                        A      1.25          1.9       149,100                                                                              13,400                                  B      5.30          6.0       16,200 6,600                                   C      7.21          23.0      53,000 5,800                                   D      24.10         29.0      66,700 7,500                                   ______________________________________                                    

Tumor and normal brain specimens were counted overnight in thegamma-well counter. All samples and standards were decay corrected forconvenience to the mid-count of the first sample in the sequence.

Conclusion

The preferential localization of radioactivity in tumor as compared tonormal tissue is demonstrated above.

I claim:
 1. A method for diagnosing malignant tumors which comprises:(a)dividing a sample of body fluid into a first and second aliquot; (b)contacting said first aliquot with a complex of Malignin and an inertcarrier for about two hours to allow protein to bind to said complex;(c) separating from said first aliquot said complex and the proteinbound to it; (d) cleaving the bound protein from said complex; (e)quantitatively determining the amount of protein cleaved from saidcomplex; (f) contacting said second aliquot with a complex of Maligninand an inert carrier for about 10 minutes to allow protein to bind tosaid complex; (g) separating from said second aliquot said complex andthe protein bound to it; (h) cleaving the bound protein from saidcomplex; (i) quantitatively determining the amount of protein cleavedfrom said complex; (j) comparing the amount of protein determined instep e, above, with that determined in step i, above; and correlatingthe difference between the two amounts with a predetermined standardvalue indicative of the tumor, wherein said Malignin is a product,derived from brain tumor cells, which forms a single line precipitatewith its specific antibody in quantitative precipitin tests and inOuchterlony gel diffusion tests, is soluble in water and aqueoussolutions having an acid or neutral pH, and insoluble at an alkaline pH,has a spectrophotometric absorption peak wave length of 280 mu, amolecular weight of about 10,000 and an amino acid compositionapproximately as follows:

    ______________________________________                                                      Approximate No.                                                               of Residues                                                     ______________________________________                                        Aspartic Acid   9                                                             Threonine       5                                                             Serine          5                                                             Glutamic Acid   13                                                            Proline         4                                                             Glycine         6                                                             Alanine         7                                                             Valine          6                                                             1/2 Cystine     1                                                             Methionine      2                                                             Isoleucine      4                                                             Leucine         8                                                             Tryosine        3                                                             Phenylalanine   3                                                             Lysine          6                                                             Histidine       2                                                             Arginine        5                                                             Approximate Total                                                                             89                                                            ______________________________________                                    

ammonia and the amino acids cysteic, hydroxyproline, norleucine,isodesmosine, hydroxylysine, lysinonorleucine and gammaaminobutyric acidbeing absent in detectable amounts.
 2. The method according to claim 1wherein said inert carrier complexed with Malignin isbromoacetylcellulose.
 3. The procedure according to claim 1 wherein thebody fluid is blood serum.
 4. The procedure according to claim 1 whereinthe bound protein is cleaved from said complex by contacting theprotein-bound complex with aqueous acetic acid.
 5. The procedureaccording to claim 1 wherein the cleaved protein is determined by aprocedure comprising reacting the protein-bound complex with aqueousacetic acid, centrifuging the mixture and determining the opticaldensity of the supernatant at about 280 m μ.
 6. The procedure of claim 2wherein the complex of Malignin and bromoacetylcellulose is suspended inphosphate buffered saline at pH 7.2 prior to being contacted with thealiquots of body fluid.
 7. The procedure of claim 6 wherein about 0.25ml of said Malignin-bromoacetylcellulose suspension comprises about100-200 micrograms of Malignin.
 8. The procedure of claim 1 wherein thebody fluid and the Malignin-carrier complex are contacted at about 4° C.